Compositions and methods for wound healing

ABSTRACT

Mice in which enhance wound healing occurs can be used to identify genes and gene products which are involved in enhanced wound healing in mammals, including humans. Methods and compositions for treating wounds, including central and peripheral nerve wounds, are also provided.

This application is a division of U.S. application Ser. No. 09/249,155 filed Feb. 12, 1999, now issued as U.S. Pat. No. 6,538,173, which claims the benefit of provisional applications Ser. No. 60/074,737 filed Feb. 13, 1998, Ser. No. 60/097,937 filed Aug. 26, 1998, and Ser. No. 60/102,051 filed Sep. 28, 1998. Each of these applications is incorporated herein in its entirety by reference.

This work was partially supported by the U.S. Government under USPHS grant AI42395 awarded by the National Institutes of Health. The U.S. Government retains certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the field of wound healing. More particularly, the invention is related to methods and compositions for enhancing wound healing in mammals.

BACKGROUND OF THE INVENTION

The biological response to tissue injury in higher organisms falls into two main categories: wound repair and regeneration (1). In amphibians, the form of wound healing seen is often epimorphic regeneration, where entire limbs can be reformed after amputation (1). In adult mammals, wound healing can involve wound repair or tissue regeneration, including the replacement of mature cells through cell proliferation (7) or replenishment of cells, but not organs, from immature stem cells (9, 11, 25). Complete wound healing, however, with perfect replacement of tissue and function, is typically not observed. Injuries to the central and peripheral nervous system, including optic nerve and spinal cord injuries, are especially refractory to healing. Thus, there is a need in the art for methods and compositions for enhancing wound healing in mammals.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions for use in healing wounds. These and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a method of identifying a gene involved in enhanced wound healing. DNA microsatellite markers which can distinguish a first and a second mouse strain are identified. The first mouse strain is a healer mouse strain, and the second mouse strain is not a healer mouse strain. Microsatellite markers which segregate with enhanced wound healing in progeny of the first and second mouse strains are identified. A chromosomal locus which contains at least one gene involved in enhanced wound healing is thereby identified.

Still another embodiment of the invention is a method of treating a wound in a mammal. A reagent which specifically binds to an expression product of a gene whose expression is decreased in a healer mouse relative to a non-healer mouse is administered to a mammal with a wound. Expression of the gene is thereby decreased.

Even another embodiment of the invention is a method of treating a wound in a mammal. An expression product of a gene whose expression is increased after wounding in a healer mouse relative to expression of the gene after wounding in a non-healer mouse is administered to a mammal with a wound. The level of the expression product in the wound is thereby increased.

Yet another embodiment of the invention is a method of restoring function after nerve injury in a mammal. A reagent which specifically binds to an expression product of a gene whose expression is decreased after wounding in a healer mouse relative to expression of the gene after wounding in a non-healer mouse is administered to a mammal with a nerve injury. Expression of the gene is thereby decreased.

Another embodiment of the invention is a method of restoring function after nerve injury in a mammal. An expression product of a gene whose expression is increased after wounding in a healer mouse relative to expression of the gene after wounding in a non-healer mouse is administered to a mammal with a nerve injury. The level of the expression product in the wound is thereby increased.

Still another embodiment of the invention is a method of treating a wound in a mammal. A cell or cellular extract obtained from a healer mouse is administered to a mammal with a wound. Healing of the wound in the mammal is thereby enhanced.

Yet another embodiment of the invention is a method of treating a wound in a mammal. A cell in which expression of a wound healing gene has been altered is administered to a mammal with a wound. Healing of the wound in the mammal is thereby enhanced.

Even another embodiment of the invention is a healer mouse having at least one quantitative trait locus selected from the group consisting of the quantitative trait loci shown in Tables 2, 9 and 16. The healer mouse exhibits an enhanced healing response to a wound compared to a mouse which does not have the at least one chromosomal locus. The healer mouse is not an MRL mouse.

Yet another embodiment of the invention is a preparation comprising a fraction of an extract of a tissue of a healer mouse. The preparation alters a biological property of a model of wound healing.

Still another embodiment of the invention is a preparation comprising cells of a healer mouse. The preparation alters a biological property of a model of wound healing.

Another embodiment of the invention is a method of identifying a factor involved in enhanced wound healing. A model of wound healing is contacted with a preparation selected from the group consisting of serum, a fraction of serum, an extract of at least one healer mouse tissue, and a fraction of an extract of at least one healer mouse tissue. A property of the model of wound healing is assayed. A preparation which alters the property of the model of wound healing is identified as comprising a factor involved in enhanced wound healing.

Yet another embodiment of the invention is a method of screening test compounds for the ability to enhance wound healing. A healer model of wound healing is contacted with a test compound. The healer model comprises cells of a healer mouse. The effect of the test compound on a biological property associated with wound healing is measured in the healer model. A test compound which enhances the biological property of the healer model is identified as a potential factor for enhancing wound healing.

The present invention thus provides the art with a mammalian model of enhanced wound healing. The healer mouse described herein can be used, inter alia, to identify genes and gene products involved in enhanced wound healing and to provide methods and compositions for healing wounds, particularly wounds of the nervous system, in mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The kinetics of ear punch hole closure. Two mm holes were punched in ears on day 0 and, for each strain of mouse, holes were measured at days indicated on the horizontal axis. Average hole diameters are shown (n=4).

FIG. 2. Photographs of the healing ear wounds. C57BL/6 (left side) and MRL/lpr (right side) ears were punched bilaterally in the center of the ears creating a 2 mm through-and-through wound and followed for 33 days. The ear holes of the C57BL/6 mouse remained open 1 day (FIG. 2A), 9 days (FIG. 2B), 20 days (FIG. 2C), and 33 days (FIG. 2D) after punching. In the MRL/lpr mouse, one can see the progression of hole closure from day 1 (FIG. 2E), day 9 (FIG. 2F), day 20 (FIG. 2G), to day 33 (FIG. 2H).

FIG. 3. Histological examination of early events in ear hole closure. The first two days after wounding of C57BL/6 ears (FIGS. 3A and 3D) and MRL/+ (FIGS. 3B and 3E) and MRL/lpr ears (FIGS. 3C and 3F) were examined. Day 1: these sections show more swelling at the wound site for the MRL tissue (smaller arrows). Eschar and inflammatory cellular infiltrate at the wound margin is similar for both strains and re-epithelialization is not seen (larger arrows). Day 2: The C57BL/6 wound is partially covered (arrow) by eschar with migrating epithelium not yet covering the wound (9 out of 14 edges examined did not close) while the MRL wound is completely covered (arrows) with epithelium (10 out of 14 edges examined completely closed). Magnification=40×; tissue is stained with hematoxylin and eosin.

FIG. 4. Day 5, 10, and 20 wounds. For all days indicated for C57BL/6 tissue (A), MRL/+ tissue (B) and MRL/lpr tissue (C). All sections are stained with hematoxylin and eosin (pictures are representative of 4 edges examined). FIG. 4A. Day 5 (A, B, C: 40×-upper panels and 20×-lower panels): Swelling at the MRL wound site is extensive with neovascularization (40×-arrows) and marked dermal fibroblast proliferation (20×-large arrows). Adnexae in the new epithelium can be seen here (20×-small arrows). A C57BL/6 wound that has failed to epithelialize is shown here, although this is not typical (20×−*). FIG. 4B. Day 10 (A, B, C: 40×-upper panels and 10×-lower panels): There is marked neovascularization (40×-arrows) and fibroblast proliferation (10×-arrows) seen in the MRL wound extending out beyond the borders of the wound where the cut cartilage edges are seen. Note the extent to which dermal cells have migrated out beyond the wound margin marked by the cartilage edge for the two MRL ears compared to the C57BL/6 ear. FIG. 4C. Day 20 (A, C:20×; B:40×-upper panels and 5×-lower panels): The prominent proliferation of fibroblasts in the dermis and the appearance of a blastema-like structure has led to significant closure of the MRL wound as originally marked by the cut edges of cartilage at the right and left margins of the photograph (5×-between arrows). By comparison, there is little extension of C57BL/6 tissue into the wound space. The homogeneity of fibroblast proliferation and ECM deposition is most striking in MRL (B:40×-upper panel).

FIG. 5. Late-stage ear wound closure. Wound sites (n=3) 81 days after wounding for C57BIL/6 (top) and MRL/lpr (bottom) mice were aligned using a dissection microscope and sutured together to assure sectioning through the former wound for MRLApr tissue. Cartilagenous islands (small arrow) present throughout the MRL/lpr section are surrounded by prominent adipocytes (large arrow). These features are absent from the C57BL/6 tissue. The tissue sections are stained with Gomori trichrome. Magnification 10×.

FIG. 6. The analysis of frequencies of wound closure on day 30 in parental and crossbred populations. Histograms of day 30 ear punch hole diameters can be seen for the following genotypes: C57BL/6 and MRL/lpr parental mice and F₁ mice (upper panel) and the first backcross to each parental strain (lower panel).

FIG. 7. Histogram of wound closure in (MRL/lpr×C57BL/6)F, and F₂ intercross populations.

FIG. 8. Additive effects of heal1 and heal3 on wound closure. Average residual wound diameters are plotted for each genotype, with the results grouped by D13Mit129 and D8Mit211. The mean of all F₂ mice±1 s.e.m. is depicted as a horizontal line in this graph. B, H, and S designate mice homozygous for the heal^(b/b)allele from C57BL/6, heterozygous for the heal^(b/s) alleles from C57BL/6 and MRL/lpr, or homozygous for the heal^(s/s) allele from MRL/lpr, respectively.

FIG. 9. Healer and non-healer mice 1.5 months after transection of the left optic nerve. FIG. 9A. The non-healer (B6) mouse exhibits complete atrophy of the left eye with marked sinking of the left eye socket compared to the normal untreated right eye. FIG. 9B. The healer (MRL) mouse exhibits only slight sinking of the left eye compared to the normal, untreated right eye.

FIG. 10. Images depicting histological longitudinal sections of the healer (MRL) mouse uncut right eye-specific tissue. These serial sections were stained with hematoxylin and eosin.

FIG. 11. A series of images depicting histological longitudinal sections of the healer (MRL) mouse cut left eye-specific tissue. These serial sections were stained with hematoxylin and eosin.

FIG. 12. FIG. 12A is an image depicting a histological longitudinal section of the healer (MRL) mouse normal uncut right eye-specific tissue. This section was stained with Bodian's silver stain specific for axons. FIG. 12B is an image depicting a histological longitudinal section of the healer (MRL) mouse cut left eye-specific tissue. This section was stained with Bodian's silver stain specific for axons. The blue arrows indicate nerve fibers and the red arrows point to oligodendrocytes.

FIG. 13. Graph depicting nerve regeneration in various strains of mice. SFI, Sensory Function Index.

FIG. 14. Graph depicting healing of ear holes in LG mice.

FIG. 15. Graph depicting adoptive transfer of the healing ability of MRL mice, wherein macrophages obtained from healer mice were transferred to non-healer mice.

FIG. 16. Results from microarray analysis of gene expression in MRL mice after ear punch. Series 1, no ear punch; Series 2, 24 hours after ear punch; Series 3, 40 hours after ear punch. Names of genes are given in Table 11.

FIG. 17. Graph showing the effect of age on the time course of ear hole closure in healer mice.

FIG. 18. Graph demonstrating that T cell depletion leads to complete healing in aged healer mice.

FIG. 19. Bar graph demonstrating that T cell receptor knock-outs show enhanced healing.

FIG. 20. Bar graph showing that adoptive transfer of fetal liver into X-irradiated non-healer recipients enhances wound healing.

FIG. 21. Two-day old explant cultures of punched ears of healer and non-healer mice. FIG. 21A, explant of a healer mouse ear punch. FIG. 21B, magnified view of the healer mouse ear punch explant. FIG. 21C, explant of a non-healer mouse ear punch. FIG. 21D, magnified view of the non-healer ear punch explant.

FIG. 22. Magnified view of the area around a healer mouse ear punch explant, demonstrating migration of cells away from the explant.

FIG. 23. Graph demonstrating that adoptive transfer of bone marrow-derived dendritic cells into ear-punched C57BL/6 Rag^(−/−) immunodeficient mice enhances wound healing.

DETAILED DESCRIPTION

It is a discovery of the present invention that healer mice, including MRL mice and strains derived from MRL mice, can be used as murine models of wound healing, particularly of the central and peripheral nervous system. These models are useful for identifying genes and gene products which are involved in enhanced wound healing in mammals, including humans, as well as for developing therapeutic compounds and methods to enhance wound healing.

Healer Mice

Typically, holes punched in the ears of non-healer mice are permanent and exhibit at most about 25% closure even 60 days after punching. Patterns of such holes can thus be used to distinguish individual non-healer mice from one another, which is a standard laboratory method for animal identification. In contrast, a hole punched in the ear of a healer mouse will close at least 70%, more preferably at least 80% or 90%, within 60 days, more preferably within 45 or 30 days. Most preferably, an ear hole of a healer mouse closes completely.

The percent closure of a hole punched in the ear of a mouse can be determined by measuring the initial diameter of the hole and by taking subsequent measurements during the first thirty days after punching. Typically, a 2 mm hole is punched. In particularly preferred healer mouse strains, complete ear hole closure occurs 30 days after punching, without scar tissue and with regeneration (perfect replacement) of cell types initially present in the ear, such as cartilage, dermis, epidermis, and blood vessels.

The healing capacity of a mouse strain can easily be tested by an ear punch assay, as described above. However, healing capacity can be assessed following other types of wounds in the mouse, including, but not limited to, digit cutting, tail cutting, and cutting or crushing of a nerve, particularly an optic or sciatic nerve, or a partial or complete cutting or crushing of the spinal cord. Liver regeneration can also be measured.

In addition to ear hole closure as described above, a mouse strain with a healer phenotype, in contrast to a non-healer mouse, may also exhibit one or more of the following characteristics. All such aspects of the healer phenotype, including enhanced tissue regeneration, are components of enhanced wound healing. Blastemas may form in the vicinity of a cut in the ear, digits, tail, or liver in a strain of healer mouse. Following injury to the liver, a strain of healer mouse may exhibit rapid replacement of liver mass and homeostasis. A healer mouse strain may exhibit breakdown in the extracellular matrix-basement membrane to allow epithelial-mesodermal interaction and may express forms of extracellular matrix-basement membrane components, such as tenascin, which are typically expressed only during development or regeneration. Organ regeneration can also occur.

Healer mouse strains may also exhibit rapid recovery from central or peripheral nerve damage, such as sciatic or optic nerve crush, or cutting or crushing of the spinal cord, and can thus be used as models in which to study regeneration of injured nerves. Nerve regeneration can be detected using functional assays appropriate for the particular nerve involved, such as electrical stimulation of the nerve and detection of contraction of the reinnervated muscle, or can be detected by observing restoration of normal neuronal architecture as a precursor to reacquisition of complete or partial normal function. In preferred healer mouse strains, regrowth and connection occurs after complete transection of nerves of the central or peripheral nervous system. Most preferably, healer mice exhibit maintenance of the optic nerve and eye with no accompanying degeneration of either structure following injury, such as crushing or transection, of the optic nerve. In addition, neurites regrow in the proper direction through a cut region of the optic nerve, and glial cells, such as oligodendrocytes, reappear in the injured area. After cutting or crushing of the spinal cord at the thoracic level, for example, preferred strains of healer mice recover at least partial function in their hind limbs and tail.

Healer mouse strains can be naturally occurring or can be generated, for example, by traditional genetic crossings, by transgenic manipulation, or by mutagenic techniques, as is known in the art. One strain of healer mouse is the MRL mouse. The MRL mouse (H-2k) is derived from an interbreeding of the LG mouse (75%; H-2d/f), the AKR mouse (12.6%; H-2k); the C3H mouse (12.1%h-2k) and the C57Bl/6 mouse (0.3%; H-2b) (13) and was selected originally for its large size. A mutant derived from this colony, MRL/lpr showed enlarged spleen and lymph nodes with age, lymphoproliferation with aberrant control of apoptosis in germinal centers, and a high susceptibility to autoimmune disease with autoantibodies, an arthritis-like syndrome, and glomerulonephritis. This was shown to be the direct result of a retrotransposon insertion into the second intron of the fas gene in the lpr strain (16, 17, 23). However, the rapid and complete wound closure described here is unrelated to fas since the MRL/+ mouse has the same healing characteristics. Furthermore, wound closure is unlinked to the lympadenopathy (R=0.4) associated with lpr mice and the autoantibodies made to histone proteins by these animals (13-15, 24). This lack of fas involvement has been confirmed in mapping studies using MRL/lpr and C57BL/6 backcross mice showing a clear genetic basis for this regeneration trait, unlinked to the fas genetic locus (McBrearty et al., Proc. Natl. Acad. Sci. U.S.A. 95, 11792-97, 1998). One characteristic of the MRL mouse is its large size, but there is no evidence that this trait is linked to adult body weight (R=0.12).

Other preferred strains of healer mice can be generated by crossing a healer mouse, such as an MRL mouse, with a non-healer mouse, such as a C57BL/6 mouse, and selecting progeny (F₁) mice which display a healer phenotype. F₁ healer mice can be intercrossed to form an F₂ generation, in which mice with a healer phenotype can be identified. Alternatively, a male or female F₁ healer mouse can be back-crossed with a female or male mouse of its healer or non-healer parent strain. The progeny of any of these crosses which display a partial or a complete healer phenotype are healer mice according to the invention. Other non-healer mouse strains, such as 129, can be crossed with a non-healer strain such as C57BL/6 to form healer mouse strains. Characteristics of the healer phenotype are those which are described above.

Mutagenic techniques, including but not limited to, targeted and non-targeted chemical mutagenesis using agents such as DMBA and ENU, as well as irradiation, for example with UV light or X-rays, can be used to induce mutations in one or more genes involved in enhanced wound healing to form a healer phenotype. Genes which can be mutated include, but are not limited to, the genes disclosed herein in Tables 4-11, 14, 15, and genes comprising the SAGE tags disclosed in Tables 12 and 13, as well as genes which regulate them.

It is also possible to create healer mouse strains using transgenic manipulations, to create transgenic, knock-in, or knock-out mice in which the function of one or more genes involved in the enhanced healing response is altered to achieve a healer phenotype. The genes of Tables 4-11, 14, 15, and genes comprising the SAGE tags disclosed in Tables 12 and 13, as well as genes which regulate them, are candidates for such manipulation. Conditional knock-in or knock-out mice, which will express one or more wound healing genes at a designated developmental stage or under particular environmental conditions, can also be constructed. Methods of creating transgenic, knock-in, and knock-out mice are well-known in the art. (See, e.g., U.S. Pat. Nos. 5,464,764 and 4,873,191).

Identification of Wound Healing Factors

Healer mice can also be used to identify factors which promote wound healing, particularly recovery from central or peripheral nerve injury, as well as genes which encode the factors. Either in vitro or in vivo models of wound healing can be used for this purpose. In vitro models can comprise tissue explants or cells. For example, all or a portion of an ear comprising a cut or a punch wound can conveniently be maintained as an explant in a collagen gel. Explants of other tissues, such as skin and nerve tissue, can also be used. Methods of maintaining tissue explants are well known in the art.

Mammalian cell lines or primary cultures of mammalian cells can also be used as in vitro models of wound healing. Suitable cell types include, but are not limited to, epidermal, mesodermal, cartilage, muscle, neuronal, glial, macrophage, and liver cells or cell lines. Many such cell lines can be obtained from commercial sources such as the American Type Culture Collection. Primary cells can be isolated from mammals such as mice, rabbits, rats, pigs, hamsters, monkeys, or humans. Methods of maintaining such cells in vitro as monolayers, suspension cultures, or cellular reaggregates are also well known.

Alternatively, the model can be a mammal, such as a rat or mouse, which has a wound. The wound can be, for example, a cut or abrasion in the skin, a tail or ear cut or an ear punch, a cut in the liver, or a severed or crushed nerve, including an optic nerve or spinal cord.

The effects of partially or fully purified proteins or nucleic acids, whole cells, such as macrophages or fibroblasts, or tissue extracts, including serum, obtained from healer mice can be tested in the in vitro or in vivo model of wound healing. Regeneration of the tissues and cells and/or morphological or biochemical changes associated with wound healing can be assessed and compared with those processes in the absence of added proteins, cells, or tissue extracts obtained from healer mice or in the presence of proteins, cells, or extracts obtained from non-healer mice. Cells such as fibroblasts, macrophages, and nerve cells isolated from healer and non-healer mice can be treated with cells, serum, or cell extracts from either non-healer or from healer mice.

Properties of a wound healing model which can be assessed include, but are not limited to, enhanced wound healing, enhanced tissue regeneration, cell growth, apoptosis, cell replication, cell movement, cell adhesion, DNA synthesis, protein synthesis, mRNA synthesis, and mRNA stability. Methods of assessing these properties include morphological assessment, either with or without the aid of a microscope, as well as biochemical and molecular biology methods well-known in the art. The alteration of at least one of these properties or of another property associated with enhanced wound healing or tissue regeneration, including an alteration in the time course of an effect, identifies a factor involved in these processes.

Extracts can be prepared from tissues and cells of healer mice using standard tissue disruption techniques, such as sonication, passage through a French press, Dounce homogenization, blending, Polytron disruption, enzymatic digestion, or blending with glass beads, followed by centrifugation. If desired, an extract can be divided into one or more fractions to further identify particular factors involved in enhanced wound healing. Any known method of fractionation, including fractional precipitation with ammonium sulfate, polyethylene glycol, or organic solvents, gel permeation or gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, dye-ligand affinity chromatography, bio-ligand affinity chromatography, high performance liquid chromatography, fast-flow liquid chromatography, electrophoretic separation methods, and the like, can be used, as is convenient.

Extracts or fractions of extracts which cause an alteration in an in vitro model or which enhance wound healing in an explant or an in vivo model can be purified by biochemical, molecular biological, and/or immunological means using standard technology or can be identified, for example, by selective inactivation. Methods such as two-dimensional gel electrophoresis can also be used to identify proteins which are present or absent, or which are present in different amounts in healer mice compared with non-healer mice. The effects of a purified factor can be confirmed using either an in vitro or an in vivo model of wound healing, as described above.

Alternatively, a subtractive hybridization-type approach can be used to identify factors capable of enhancing wound healing or tissue can be obtained from healer mice, and mRNA can be extracted from these cells using methods well known in the art. Specific mRNAs which are expressed in the cells of healer mice can be isolated by subtractive hybridization of healer mRNA with mRNA obtained from identical cells from non-healer mice. Differentially expressed mRNAs can then be reverse transcribed to form cDNA.

Genes encoding such factors may then be identified, cloned, sequenced and otherwise characterized, as is known in the art. cDNA can be cloned into an expression vector using standard methodologies. Protein expressed from the cDNA can be tested for the ability to enhance wound healing or tissue regeneration of mammalian tissue in an in vitro or in vivo model such as those described above. cDNA which encodes a protein identified as involved in enhanced wound healing can be sequenced and further characterized as desired using ordinary molecular biology technology. The technology required to perform these experiments is well known in the art and is described, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, New York, 1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Green & Wiley, New York, 1993).

Any murine wound healing factors which are identified using a healer mouse can be used to identify counterpart factors in other mammals, particularly in humans. For example, degenerate primers encoding portions of murine wound healing factors can be constructed and used to probe cDNA libraries of other mammalian species, preferably human cDNA libraries. Antibodies which specifically bind to a murine wound healing factor can be used to identify similar factors in cells, tissues, extracts, or expression libraries of other mammalian species, preferably human. Thus, the invention should be construed to include the identification of any and all mammalian wound healing factors which share homology with those identified in the murine systems described herein.

The invention also provides methods of screening test compounds for the ability to enhance wound healing. The test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. Members of combinatorial libraries can also be screened for wound healing activity.

A healer model of wound healing is contacted with a test compound. The healer model comprises cells of a healer mouse, and can be any of the in vitro or in vivo types of wound healing models described above. Healer models in which healer and non-healer cells are mixed can also be used. The effect of the test compound on a biological property associated with wound healing is measured in the healer model. These biological properties include, but are not limited to, enhanced wound healing, enhanced tissue regeneration, cell growth, apoptosis, cell replication, cell movement, cell adhesion, DNA synthesis, protein synthesis, mRNA synthesis, and mRNA stability. A test compound which enhances the biological property of the healer model is identified as a potential factor for enhancing wound healing.

Optionally, the effect of the test compound on the biological property of the healer model can be compared with its effect on the same or a similar biological property of a non-healer model of wound healing. Non-healer models are any of the in vitro or in vivo models described above, but do not comprise healer mouse cells. A test compound which alters the biological property of the non-healer model such that it resembles the biological property of the healer model is identified as a potential factor for enhancing wound healing.

Identification of Healer Chromosomal Loci and Genes

Healer mice are particularly useful for identifying genes which are differentially expressed in healer vs. non-healer mice and which are involved in enhanced wound healing. Wound healing genes include genes whose products are directly involved in enhanced wound healing or tissue regeneration, as well as genes which regulate them. Such genes can be identified by a variety of methods. For example, differentially expressed wound healing genes can be identified based on microarray, SAGE, and RT-PCR analyses, as described in the Examples, below.

Chromosomal loci which contain at least one gene which encodes a protein involved in enhanced wound healing can be identified by genome screening. DNA microsatellite markers are identified which can distinguish two strains of mice. One of the two strains is a healer mouse strain, such as the MRL mouse or its progeny. The other strain is a non-healer mouse, such as a C57BL/6. A number of suitable mouse strains are available commercially, for example from Jackson or Taconic Laboratories. Microsatellite markers which segregate with enhanced wound healing in progeny of the two strains identify a chromosomal locus which contains at least one gene which is involved in enhanced wound healing. The product of this gene may be directly involved in enhanced wound healing or may regulate expression of another gene which is involved in enhanced wound healing. This method is described in detail in Examples 3-7, below.

Certain quantitative trait loci identified by this method can be observed to segregate with either male or female healer mice. For example, the quantitative trait loci identified on chromosome 7 segregate with male mice, as do some of the quantitative trait loci on chromosome 13. In addition, quantitative trait loci identified on chromosomes 4 and 18 segregate with male mice. On the other hand, the quantitative trait loci identified on chromosomes 12 and 15 segregate with female mice.

Congenic mouse strains, created by successive back-crossings of the F₁ generation of a healer x non-healer cross with its non-healer parent strain and selected for a healer phenotype, are especially useful for identifying chromosomal loci which segregate with the healer phenotype. The healer phenotype of such congenic healer mice ranges from mice with the ability to heal a 2 mm ear hole at least 75% to mice which heal such ear holes completely. For example, after six successive back-crossings, a healer mouse strain was identified which retains approximately 2% of the markers of the healer mouse genome, including the quantitative trait loci shown in Tables 2, 9, and 16, and which exhibits a healer phenotype (Example 19 and Table 16). By repeated back-crossings, strains of healer mice can be created which have at least one of the quantitative trait loci shown in Tables 2, 9, and 16. One locus of particular interest is marker 39, located at 29 cM on chromosome 16. This locus coincides with the location of a wound healing gene termed p63, located between 14 and 29 cM on chromosome 16.

A number of genes involved in enhanced wound healing or tissue regeneration have been identified using these methods. Genes which are differentially expressed between healer and non-healer mouse dendritic cells are shown in Table 15. Genes whose expression is differentially increased or decreased in healer mice after wounding, such as ear punching, are shown in Tables 5, 6, 7, 8, 10, 11, and 15. Levels of some gene products, such as ezrin, c-Jun, and c-myc, are increased in both males and females. Levels of other gene products, such as PI-K p58, and glutathione s-transferase, are decreased in both males and females after wounding.

Still other gene products show more complex changes after wounding, being altered only in males or females, at early or late stages after wounding, or a combination of both. For example, the hox8 (msx2) gene is over-expressed in healer mice at 0, 1, and 5 days after ear punch, but its expression decreases at about 20 days after ear punch, when the ear begins to heal.

Therapeutic Methods for Treating Wounds

Manipulation of the expression of wound healing genes in non-healer mammals, including altering effective levels of their expression products, in non-healer mammals can be used to treat wounds and to enhance or promote wound healing, particularly in humans.

Wounds can be treated at early or late stages after wounding by selectively manipulating expression of particular wound healing genes. Certain of these genes are identified in Tables 5, 6, 7, 8, 10, 11, and 15. Optionally, expression of one or more wound healing genes can be manipulated simultaneously or sequentially. Furthermore, wounds in males or females can be treated most effectively by selecting the appropriate wound healing genes for manipulation.

Expression of genes of Tables 5, 6, 7, 8, 10, 11, and 15 whose expression is selectively decreased and/or increased in healer mice at particular times after wounding can be manipulated in order to enhance wound healing. Particularly preferred genes are msx2 and RARg. Any method known in the art can be used to decrease or increase expression of a wound healing gene. Methods of decreasing effective expression of a wound healing gene include, but are not limited to, use of ribozyme, antisense, or antibody technologies. Methods of increasing effective expression of a wound healing gene include, but are not limited to, providing polynucleotide sequences encoding expression products of the gene such as protein or mRNA, as well as providing the expression products themselves.

In one embodiment of the invention, expression of a wound healing gene is altered using an antisense oligonucleotide sequence. The antisense sequence is complementary to at least a portion of the coding sequence of the gene. The coding sequences of preferred wound healing genes can be obtained from databases such as Genbank; accession numbers for some of thtese genes are provided in Table 14. Preferably, the antisense oligonucleotide sequence is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences can also be used. Antisense oligonucleotide molecules can be provided in a construct and introduced into cells using standard methodologies to decrease expression of one or more wound healing genes.

Antibodies which specifically bind to a wound healing protein can also be used to alter the effective expression levels of a wound healing gene. Wound healing-specific antibodies bind to a wound healing protein and prevent the protein from functioning in the cell. Preparations of polyclonal and monoclonal antibodies can be made using standard methods. Antibody fragments such as Fab, single-chain Fv, or F(ab′)₂ fragments can also be prepared. If desired, antibodies and antibody fragments can also be “humanized” in order to prevent a patient from mounting an immune response against the antibody when it is used therapeutically, as is known in the art. Other types of antibodies, such as chimeric antibodies, can be constructed as disclosed, for example, in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, can be prepared and used in methods of the invention. Anti-idiotype antibodies, directed against unique sequence variants in wound healing gene products, can also be used in therapeutic methods of the invention.

Useful antibodies specifically bind to epitopes present in proteins encoded by a wound healing gene. The amino acid sequences of these proteins are available from Genbank (see, e.g., Table 14 for accession numbers). Antibodies which specifically bind to wound healing proteins provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies which specifically bind to a wound healing protein do not detect other proteins in immunochemical assays and can immunoprecipitate the wound healing protein from solution.

Antibodies can be purified by methods well known in the art. For example, the antibodies are affinity purified, by passing the antibodies over a column to which a wound healing protein is bound. The bound antibodies can then be eluted from the column, for example, using a buffer with a high salt concentration.

Ribozymes can be used to inhibit gene function by hybridizing to and cleaving an RNA sequence of a wound healing mRNA, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The nucleotide sequences of wound healing genes, available from databases such as Genbank (see Table 14), are sources of suitable hybridization region sequences.

Preferably, the mechanism used to decrease the effective expression of the involved gene decreases levels of gene expression products, such as mRNA or protein, by 50%, 60%, 70%, or 80%. Most preferably, effective expression of the gene is decreased by 90%, 95%, 99%, or 100%. Effective expression of the involved gene can be assessed using methods well known in the art, such as hybridization of nucleotide probes to mRNA of a wound healing gene, quantitative RT-PCR, or detection of wound healing protein using specific antibodies.

Expression of wound healing genes whose expression is increased in healer mice after wounding can be effectively increased in a non-healer mammal in order to enhance wound healing. Any method known in the art for enhancing gene expression can be used for this purpose. For example, the coding sequence of one or more wound healing genes can be delivered to cells in the vicinity of a wound. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce the coding sequence into cells in which it is desired to increase wound healing gene expression. Alternatively, if it is desired that the cells stably retain the construct, it can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. The construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of the coding sequence in the cells.

Vectors, such as retroviral- or adenoviral-based vectors, can be used for this purpose. Recombinant retroviruses are described in numerous references, including Mann et al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, and U.S. Pat. Nos. 4,405,712, 4,861,719, and 4,980,289.

Advenoviral vectors are preferred. The use of adenoviral vectors in vitro is described in Chatterjee et al., Science 258: 1485-1488 (1992), Walsh et al., Proc. Nat'l. Acad. Sci. 89: 7257-7261 (1992), Walsh et al., J. Clin. Invest. 94: 1440-1448 (1994), Flotte et al., J. Biol. Chem. 268: 3781-3790 (1993), Ponnazhagan et al., J. Exp. Med. 179: 733-738 (1994), Miller et al., Proc. Nat'l Acad. Sci. 91: 10183-10187 (1994), Einerhand et al., Gene Ther. 2: 336-343 (1995), Luo et al., Exp. Hematol. 23: 1261-1267 (1995), and Zhou et al., Gene Therapy 3: 223-229 (1996). In vivo use of adenoviral vectors is described in Flotte et al., Proc. Nat'l Acad. Sci. 90: 10613-10617 (1993), and Kaplitt et al., Nature Genet. 8:148-153 (1994). Other viral vectors, such as those based on togaviruses or alpha viruses, can also be used.

A wound healing coding sequence can also be combined with a condensing agent, such as polylysine, polyarginine, polyornithine, protamine, spermine, spermidine, or putrescine, to form a gene delivery vehicle. Many suitable methods for making such linkages are known in the art. Alternatively, a wound healing coding sequence can be associated with a liposome to deliver the coding sequence to cells at the site of the wound. Other suitable methods of providing wound healing coding sequences include DNA-ligand combinations, such as those disclosed in Wu et al., J. Biol. Chem. 264:16985-16987 (1989). Wound healing coding sequences can also be delivered to the site of an internal wound, for example, using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991). Expression of a wound healing gene can be monitored by detecting production of mRNA which hybridizes to a delivered coding sequence or by detecting the protein product of the gene using, for example, immunological techniques.

Expression of an endogenous wound healing gene in a cell can also be altered by introducing in frame with the endogenous gene a DNA construct comprising a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site by homologous recombination, such that a homologously recombinant cell comprising a new transcription unit is formed. The new transcription unit can be used to turn the wound healing gene on or off as desired. This method of affecting endogenous gene expression is taught in U.S. Pat. No. 5,641,670, which is incorporated herein by reference.

The targeting sequence is a segment of at least 10, 15, 20, or 50 contiguous nucleotides selected from the nucleotide sequence of a wound healing gene. The nucleotide sequences of the wound healing genes disclosed herein are available, for example, from Genbank, using the accession numbers provided in Table 14. The transcription unitlis located upstream of a coding sequence of the endogenous wound healing gene. The exogenous regulatory sequence directs transcription of the coding sequence of the wound healing gene.

Optionally, effective expression of one or more wound healing genes can be altered in cells which have been removed from a mammal, such as dermal fibroblasts or peripheral blood leukocytes. The cells can then be replaced into the same or another mammal with a wound, preferably to or within the vicinity of the wound to enhance healing of the wound. Alternatively, cells obtained from healer mice can be used to treat a wound in a mammal. Preferred cells include macrophages, stem cells, fetal liver cells, peripheral blood leukocytes, and bone marrow cells. Extracts from these cells can be prepared using standard methodologies and used for wound treatment. The cells or cellular extracts can be placed directly at the site of the wound to promote its healing.

In another embodiment, protein products of wound healing genes whose expression is increased after wounding in healer mice can be applied directly to the area of the wound. Protein products of wound healing genes can be used in a pharmaceutically acceptable composition and can be applied topically, as is well known in the art and described below.

The lowered capacity for wound healing which occurs in aged mammals, including humans, can be enhanced by suppressing T cell function, for example using an antibody which specifically binds to a T cell receptor (TCR) or, in experimental mammals, by genetically eliminating functional T cell receptors. Anti-TCR antibodies are available in the art or can be prepared using established methodologies. Optionally, a polynucleotide encoding a single chain anti-TCR antibody can be used. Anti-TCR antibodies can be administered to mammals, including humans, to reduce levels of functional TCRs. Sub-populations of TCRs can also be targeted.

Wounds which can be treated using methods of the invention include, but are not limited to, cuts, stretches, tears, pulls, abrasions, burns, bone breaks, crushes, scrapes, contusions, bruises, and the like. Particularly, peripheral or central nerve injuries, such as crushed or severed nerves, including the spinal cord, can be treated. Methods and compositions of the invention can be used to treat and thus enhance healing of a wound by promoting processes such as angiogenesis, chondrogenesis, return of hair follicles and/or sebaceous glands, reepithelialization, rapid connective tissue proliferation, deposition of organized extracellular matrix, and restoration of normal tissue architecture and function. Surgical adhesions can be prevented by prophylactic treatment of surgical incisions using compositions and methods of the invention. These methods and compositions are useful in any situation in which regeneration or healing of a wound without formation of scar tissue is desired.

Wound healing compositions of the invention can comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those in the art. Such carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts can also be used in wound healing compositions, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. Wound healing compositions can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes, such as those described in U.S. Pat. No. 5,422,120, WO 95/13796, WO 91/14445, or EP 524,968 B 1, can also be used as a carrier for a wound healing composition.

Administration of wound healing compositions of the invention can include local or systemic administration, including injection, oral administration, particle gun, or catheterized administration, and topical administration. For treatment of wounds on the surface of the body, a wound healing composition is typically prepared in a topical form, either as a liquid solution, suspension, gel, or cream. However, solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared, for local treatment of internal wounds. Both the dose of a particular wound healing composition and the means of administering the composition can be determined based on specific qualities of the wound healing composition, the condition, age, and weight of the patient, the type and extent of the wound being treated, and other relevant factors.

The above disclosure generally describes the present invention. All references cited in this disclosure are expressly incorporated herein by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

Materials and Methods

The following materials and methods were used in the examples described below.

Mice. The MRL/MpJ-Fas^(lpr) (hereafter referred to as MRL/lpr) mice were obtained from the Jackson Laboratory (Bar Harbor, Me.). C57BL/6 mice were acquired from the Taconic Laboratory (Germantown, N.Y.). Mice were bred and maintained under standard conditions at The Wistar Institute (Philadelphia, Pa.).

F₁, F₂ and MRL/lpr backcross populations were generated to conduct the genetic studies. The female parent used for generating the F₂ and backcross animals was MRL/lpr. In the examples below, alleles derived from the C57BL/6 parent are designated B and the MRL/lpr-derived alleles are designated S.

Phenotyping A 2.1 millimeter through-and-through hole was made in the center of the cartilagenous part of both ears of six week old mice using a metal ear punch (Fisher Scientific, Pittsburgh, cat # 01-337B). The holes were measured at the time of wounding and followed for wound closure using a grid-etched reticle (Bausch and Lomb, 7×).

Histology. Ears were removed with scissors by cutting at the base of the pinna. They were fixed overnight in 10% buffered formalin. To facilitate sectioning, they were held flat during fixation by inverting the lid of a processing cassette on the base and sandwiching the flattened ear by applying gentle pressure with a rubber band.

Once fixed, ears were bisected across the widest point of the hole under a dissecting microscope, using a #10 scalpel blade. The two halves were then glued together with collodion, again using the dissecting microscope to obtain perfect alignment of the cut edges and hole margins. Because the collodion would most likely dissolve in reagents used to prepare specimens for paraffin embedding, the ears were sutured together using 5.0 silk on a 1½ inch straight Keith Abdominal cutting needle with a triangular point.

Tissues were embedded in paraffin and sectioned so that the cut edges containing the hole margins were in the plane of the section. Sections were stained with hematoxylin and eosin or with Gomori trichrome stain.

Genetic Analysis. Genomic DNA was prepared from the liver of each animal in the (MRL/lpr X C57BL/6)×(MRL/lpr X C57BL/6) F₂ population. Frozen tissue was homogenized and digested overnight with 100 mg/ml proteinase K. Samples were treated with two phenol:chloroform extractions and one final chloroform extraction. Finally, genomic DNA was purified by an overnight dialysis against TE buffer.

PCR primers, purchased from Research Genetics (Huntsville, Ala.), were employed to perform a genome wide scan of the mouse. Amplification was conducted using Boehringer Mannheim reagents with the following concentrations: 1×PCR buffer, 0.375 mM dNTPs, 0.5 U/μls of Taq polymerase, 0.165 μM of each primer, and 160 ng/20 μls of genomic DNA. Cycling conditions include a 1 min at 95° C. denaturing, 35 to 50 cycles of 1 min at 94° C., 1 min 30 sec at 55° C., 2 min 10 see at 72° C., and a 6 min final extension at 72° C. PCR products were resolved using 3% Metaphor agarose (FMC) and were visualized through ethidium bromide staining. This method was followed for the majority of polymorphic markers. In the case of small base pair differences, PCR amplification was carried out using ³²P-ATP labeled forward primers as described (38). Radiolabelled PCR products were then fractionated by size on 6% denaturing polyacrylamide gels.

Statistics. Genotype data was organized and analyzed through the use of Map Manager QT (39). For quantitative trait analysis, critical threshold values for significance of linkage were determined by the permutation test, based on a regression model developed by Churchill and Doerge (40, 41). The values for the additive model of inheritance were calculated in terms of a likelihood ratio statistic (LRS).

The threshold in the F₂ under assumptions of the additive model for suggestive linkage is LRS≧3.3 and for significant linkage is LRS≧10.7. The dominant, free and recessive models were also tested and did not show a significant difference (i.e., they were less than 18-fold different) in resultant p values. The additive regression model was used because it is the simplest model and because it is consistent with the mode of inheritance of the quantitative trait loci (QTL) determined in this study. It should also be noted that the use of the additive regression model does not assume the pattern of inheritance to be strictly additive and at present the degree of additivity and dominance has not been determined. The threshold values in the backcross are 3.7 and 11.8, respectively. Loci were named as healing quantitative QTL if they independently attained significance in either cross or a suggestive level of significance in one cross, confirmed in the other (p<0.05). Microsatellite markers were evaluated individually for linkage to the healing phenotype, based on the threshold values. In addition, a mean healing score for markers closely linked to each healing QTL was calculated independently of other loci using ANOVA, using a Bonferroni/Dunn post-hoc test for making the three possible pairwise comparisons in the F₂, resulting in single-locus genotypic values (Table 3) (45).

EXAMPLE 1

Kinetics and Gross Aspects of the Wound Healing Phenotype.

C57BL/6 and MRL mice were ear punched using a standard metal ear punch to create a well circumscribed circular surgical wound of about 2 mm in diameter at a site at which the thickness of the ear is considerably less than a millimeter. The animals used initially were within the 8-12 week young adult age range so that early developmental considerations would not be an issue. As can be seen in FIG. 1, by day 15 maximal closure was achieved in the C57BL/6 with a 30% reduction in the original hole diameter, which remains stable. In contrast, the MRL achieves an 85% reduction in hole diameter by day 15 with complete closure by day 25. Re-examination of the ears on day 81 showed no further changes.

In FIG. 2, the closed MRL wound is evident and it is difficult to identify the original site of the hole since there is no fibrosis or scarring.

EXAMPLE 2

Histological Aspects of the Wound Healing Phenotype.

Histologic sections of healing ear punch holes were examined during the first two days after injury to determine if there was a defect in the ability of their epithelium to migrate across the cut edge of the dermis and cartilage, thereby allowing uninhibited connective tissue proliferation (FIG. 3). However, not only did epithelium promptly migrate across the MRL wounds, but this change occurred one day earlier for MRL mice (day 2) than for C57BL/6 mice (day 3). Indeed, for MRL mice epithelium completely covered virtually all wounds examined after day 1. Epithelium covered all C57BL/6 wounds examined after and including day 3, except for one wound from day 5 (FIG. 4A) which showed continued presence of eschar with migrating epithelium failing to bridge the cut edge.

As ears were prepared for histology, two grossly observable differences were noted between C57BL/6 and MRL. First, for all time points, the tissue surrounding the wounds in the MRL ears was severely hyperemic when compared to that of the C57BL/6 wounds. Second, starting on day 4, and continuing on each succeeding day, a prominent annular swelling was observed around the MRL wounds that was absent for C57BL/6 wounds.

Consistent with the grossly observable differences between the wounds, histological examination at each timepoint (5 days, FIG. 4A; 10 days, FIG. 4B; 20 days, FIG. 4C) showed a marked difference in the degree of angiogenesis, cell proliferation, connective tissue matrix formation, fibroblast migration, and ECM deposition which occurred in the two strains. Also, the presence of hair follicles with accompanying sebaceous glands within the healing wounds was noted for both MRL and C57BL/6 wounds but appeared more prominent and numerous in MRL than in C57BL/6 wounds.

At all of these time points, C57BL/6 wounds have shown limited progression beyond the cut cartilage margins and have a distinct paucity of epidermal hair follicles and sebaceous glands. In contrast, the MRL wounds show marked progress towards full closure due to extensive dermal proliferation and are well supplied with hair follicles and sebaceous glands in the new growth zone. ECM is laid down so as to preserve normal architecture, underlying connective tissue is hyperplastic, and the epidermis is rich and thick. The ear cartilage layer has not significantly extended into the wound site beyond the initial cut margin.

C57BL/6 and MRL ears are shown on day 81 after wounding in FIG. 5. Numerous ingrowths of cartilage can be seen in the MRL ear that are absent from the C57BL/6 ear. The cartilage ingrowths are surrounded by numerous adipocytes which normally make up the minor subcutaneous or hypodermal layer connecting ear cartilage to dermis. It is not clear why fat cells have come to be such a prominent cell type by this time point.

EXAMPLE 3

The Pattern of Inheritance of the Wound Healing Trait is Quantitative.

Our initial findings on the hereditary nature of the wound healing trait can be seen in FIG. 6 (upper panel). In these studies, mice were ear punched at 6 weeks of age and were examined at 2 weeks and 4 weeks after ear punching. The 4 week ear hole size of the MRL mice ranged from 0 to 0.4 mm, while the ear hole size of the C57BL/6 mice ranged from 1.2 to 1.6 mm. These two healing phenotypes were non-overlapping. Fifteen F₁ mice bred from MRL×C57BL/6 had ear holes intermediate between the two parents (ranging from 0.4 to 1.1 mm).

In an initial experiment, two backcross populations were created by using (MRL female×C57BL/6 male) F₁ females and mating them to the parental males (FIG. 6, lower panel). The backcross population to MRL displayed a curve skewed to MRL-type healing. In the backcross population to C57BL/6, the progeny showed a curve with its mean displaced to C57BL/6-type (i.e. poor) healing. The healing thus appears to be a quantitative trait.

EXAMPLE 4

The Pattern of Inheritance of the Wound Healing Trait.

All MRL mice quickly and effectively close the wounds in their ears; C57BL/6 (B6) mice are unable to completely close their wounds (36). The F₁ has an intermediate phenotype, with considerable variability (FIG. 7). Like the F₁, the (MRL/lpr×B6)F₂ population demonstrates a bell-shaped curve of healing diameters (FIG. 7). The backcross (BCI) population to MRL/lpr ((MRL/lpr×B6)F₁×MRL/lpr) displays a curve skewed to MRL/lpr-type healing, whereas in the backcross population to B6 [(MRL/lpr×B6)F₁×B6], the progeny show a mean displaced to B6-type (i.e. poor) healing (36).

The healing profiles of each of the populations used in this study are given in Table 1. The distribution of the variance in each of these populations compared to those of the parental and F₁ mice were used to give a rough estimate of the number of unlinked genes that contribute to this quantitative trait (42, 43). From this calculation, it is likely that at minimum three to four unlinked loci (QTLs) have an impact on the healing trait in this strain combination.

EXAMPLE 5

Mapping of SSLP Markers and Significant Threshold Values.

A total of 436 SSLP markers were tested for potential polymorphisms between the two strains of mice. Ninety two markers detected allelic variants in the parental strains and therefore were used for genotyping of the segregating populations. Markers were chosen based on their location in the genome, in an attempt to make a linkage map with an even spacing of 20 centimorgans between markers to generate a complete genome-wide scan (44). In regions where linkage to the healing trait was detected, the density of markers was increased to obtain a more accurate genetic dissection in the area of interest. Overall, genomic coverage reached ˜97.7 percent across the nineteen autosomes. Surprisingly, despite the distant genetic relationship between these two inbred strains (13, 37), no polymorphisms were found on the X chromosome (0/18 primers tested, data not shown).

The 92 polymorphic microsatellite markers were then used to map the wound healing/regeneration trait in 101 mice from the (MRL/lpr X C57BL/6)F₂ intercross. For assessment of the probability of genetic linkage, critical values were calculated from this database using the permutation test (40; see Materials and Methods). The order of all makers in this linkage analysis was consistent with the order predicted by the available genomic maps (Whitehead Institute/MIT, and Mouse Genome Database, The Jackson Laboratory, Bar Harbor, ME, available at the URL address: http file type, www host server, domain name informatics.jax.org.

EXAMPLE 6

Mapping of Quantitative Trait Loci Linked to the Healing Phenotype in the F₂ Population.

Table 2 shows all of the microsatellite markers that were positive for linkage to the healing phenotype in two crosses and Table 3 lists the healing scores for markers associated with wound closure. Table 9 shows microsatellite markers that were positive for linkage to the healing phenotype analyzed separately for male and female mice.

In the F₂ cross, QTLs that contribute to the healing phenotype and are derived from MRL/lpr exist in three primary support intervals. Two of these QTLs are located on individual sites on chromosome 13, and are designated heal2 and heal3. These QTLs are located as follows (with their corresponding microsatellite marker): heal2, on proximal chromosome 13, near D13Mit115 (p=0.0019) and heal3, at a more distal location near D13Mit129 (p=0.0010). One of the two QTLs on chromosome 13 (heal3) has achieved significant likelihood of linkage to the healing trait while the other (heal2) is suggestive, though confirmed in a second cross (see below).

Multiple markers near these loci show a suggestive level of significance, including for heal2: D13Mit135 and D13Mit116 and for heal3: D13Mit53, D13Mit151, D13Mit144, and D13Mit107 (Table 2). D13Nds1 and D13Mit191 both achieved highly suggestive LRS values but the assignment of a QTL in this region is provisional at the present time because the 95% confidence intervals for these markers and the two flanking QTLs overlap (not shown). Nevertheless, there are distinct breaks between these QTLs in the level of significance for their linkage to healing with no deviation from the predicted order of these markers. A second region that contains a QTL with significant linkage to the healing phenotype was detected on chromosome 15, in the region of marker D15Mit244 (p=0.0011). Other microsatellite markers mapping to this location which meet the criteria for suggestive linkage to wound healing include D15Mit172 and D15Mit14. We have designated this QTL heal4.

In addition to the QTL on chromosome 13 and chromosome 15, which have MRL/lpr-derived healing alleles, a B6-derived healing QTL was mapped to chromosome eight, near the marker D8Mit211, with a significant LRS value (10.7, p=0.0011). Other markers in this location which showed linkage were D8Mit132, D8Mit166, and D8Mit249. This locus was the first QTL identified and was designated heal1.

The contribution of each heal locus to the process of wound closure, as expressed by the single-locus genotypic values (45) (Table 3), generally fit an additive mode of inheritance, with the heterozygote healing score approximately halfway between the scores of the two homozygotes. One exception is the heal3 QTL, which may be recessive (i.e., heal3^(s/s) homozygotes show significantly better wound closure than either heterozygotes or heal3^(b/b) homozygotes). In addition, the heal3 QTL appears to interact with heal1 to give the most completely healed ear holes (ANOVA, p=0.017). The average residual wound in heal1^(b/b) homozygotes is 0.73+0.27; however, in animals that are both heal1^(b/b) and heal3^(s/s), residual wound size is 0.53+0.30 (FIG. 2). Conversely, in mice homozygous for heal1^(s/s) but also homozygous for heal3^(b/b), the residual wound size is 1.2+0.22. Other pairs of heal QTLs also show these largely additive interactions, but do not attain statistical significance.

EXAMPLE 7

Mapping of Quantitative Trait Loci Associated with the Healing Phenotype in the Backcross.

To confirm the linkage assignments seen in the F₂, we conducted a small backcross study (42 mice), using (MRL/lpr×B6) F₁ females and MRL/lpr males as parents. The F1 between MRL/lpr and B6 has an intermediate wound closure phenotype, and all progeny in this cross were expected to show intermediate to good healing. In fact, this was largely the case, although several mice in the backcross had poor wound healing. An analysis of this cross showed linkage to healing at locations that coincided with the supported intervals of association seen in the F₂ for both of the two QTLs on chromosome 13 (heal2 and heal3). These QTLs were near markers D13Mit115 (LRS value=5.0, p=0.0261) and D13Mit129 (LRS value=8.3, p=0.0040), respectively (Table 2). In addition, linkage to the healing phenotype was also detected on chromosome 12 (heal5) at marker D12Mit132 with a LRS value of 10.9 (p=0.0009) and at the closely-linked marker, D12Mit233 (LRS=9.4, p=0.0022). This linkage was supported by suggestive linkage in the F₂. The single locus genotypic value for residual wound diameter for the heal5 (DI2Mit132) QTL is also given in Table 3. Finally, a locus showing a highly suggestive LRS value of 10.2 (p=0.0014) was found on chromosome 7 near marker D7Mit220.

EXAMPLE 8

Differential Gene Expression in Healer and Non-Healer Mice.

Assays were carried out to analyze differential gene expression in healer versus non healer mice at different times during the healing process after ear punch.

SAGE analysis. mRNA transcripts were identified and their levels quantitated using the SAGE technique, as described in U.S. Pat. No. 5,695,937. In order to use SAGE for transcript identification and quantitation, messenger RNA (mRNA) was first prepared from the desired cell or tissue sample. Complementary DNA (cDNA) was then synthesized from the mRNA using standard techniques. The entire population of cDNA molecules was treated to create a single unique “tag” from each cDNA. These tags are listed in Tables 12 (C57BL/6) and 13 (MRL).

The sequence of the tags serves to identify each transcript. The number of times each tag occurs measures the number of copies of the mRNA originally present in the biological sample. Tags can then be identified and quantitated. Gene expression data obtained from SAGE analysis of each tissue was stored in a database to facilitate multiple comparisons with gene expression in other tissues.

Microarray analysis. Gene expression in wound tissue of healer and nonhealer mice and their progeny was analyzed using the Atlas Mouse cDNA Expression Array (Clontech). This array includes 588 genes that playkey roles in a variety of biological processes. cDNA was synthesized from healing wound tissue from healer and nonhealer mice, labeled with ³²P, and hybridized separately to the arrays according to the protocol provided. After a high-stringency wash step and autoradiography, expression profiles were obtained.

RT-PCR and Differential Display. The presence of known gene products in healing wound tissue was analyzed using RT-PCR and Differential Display analysis. cDNA was synthesized from RNA using reverse transcriptase. The cDNA was amplified using specific primers and displayed as fragments by gel electrophoresis for comparison. Quantitation was carried out using titrations of input material, and then samples were compared. This method is valuable for assessing the presence of known gene products and is relatively quantitative.

Expression of candidate genes identified in the genome screen described above, were separated into the following groups. Group I comprises genes which may be directly involved in wound healing based on their mapping to a chromosomal locus identified in the genome screen. Group IA comprises genes in loci whose correlation with wound healing are statistically suggestive. Group IB comprises genes in loci whose correlation with wound healing are statistically significant. Group II comprises genes which are regulated by products of the mapped genes in Group I.

The results of these assays are given in Tables 5-8. Differential expression of several candidate gene products were confirmed in two assays: MSX-2 or HOX8 (chromosome 13) and RARG (chromosome 15) were identified by both RT-PCR and microarray analyses at early time points. The gene encoding epidermal keratin (on chromosome 15) was identified by microarray at all time points and by SAGE analysis. Expression of RARG and epidermal keratin could be related, because retinoic acid (RA) causes growth of epidermal cells via the RA receptors in skin (i.e., RARG). Epidermal keratin would then be upregulated.

EXAMPLE 9

Quantitative Trait Loci Associated with Healing.

The Mouse Genome Database and associated literature were searched for potential candidates near the heal QTL (Table 4). The first locus, heal1, located on chromosome 8, is derived from C57BL/6 and is one of the strongest QTL of the five (p=0.0011), In the absence of contributing genes from MRL, C57BL/6 mice clearly cannot accomplish complete wound closure with heal1 alone. It has been shown that alleles that contribute to a trait from a parental strain that does not display that trait are not uncommon (46). Thus, it is not surprising that heal1 shows a strong additive effect with MRL loci in the F₂ (see FIG. 8). Candidate genes for heal1 (Table 4) in the strongest supported interval include the guanine nucleotide binding protein alpha 0, Gnao, an alpha subunit of a heterotrimeric G-protein which interacts with an activated G protein coupled receptor (Gp-cr), preceding downstream signaling (47). It is interesting to speculate that the basis of the additive interaction of heal1 with heal3 is an interaction between the gene products encoded by Gnao and the Gp-cr 18 found in the heal3 interval.

Though the method used for determining QTL cannot separate multiple loci in the same linkage group, we have observed highly suggestive LRS values for two regions of chromosome 13 in addition to the two QTLs identified (Table 2). That these may be unique QTLs is supported by the fact that they are separable from each other in phenotype congenics that have been generated (data not shown). One of these regions is located on chromosome 13, near marker D13nds1. Msx2 (also known as Hox8) is found in this interval and is expressed in regenerating amphibian tissue (48) as well as regrowing fingertips in neonatal mice (49). In this regard, we have evidence that Msx2 expression is different in healing ear tissue between MRL and B6 mice (Samulewicz et al, manuscript in preparation). This difference could be due to a polymorphism in the Msx2 gene itself, or to the indirect effect of an FGF signaling difference (50) mediated through the FGF receptor, FGFR4 (51), which is also encoded by a candidate gene located in this interval.

Heal4 is found on chromosome 15 near marker D15Mit244. The chromosome 15 QTL is strongly associated with the wound closure trait (p=0.0011) and is located in an area rich in candidate genes, including the gene encoding retinoic acid receptor gamma (Rarg), members of the keratin family which influence differentiation in the epidermis, as well as developmental genes known as homeobox and wnt genes. The retinoic acid pathway is known for its role in regeneration in amphibians (32, 34, and 52). Furthermore, the gamma subtype of the RAR displays preferential expression over the alpha and beta subtypes in skin and cartilage tissues (53), which is the site where the MRL/lpr healing trait is evaluated in the present study.

Finally, this set of genes does not include the fas gene, H-2, or any other gene known to play a role in the autoimmune profile of MRL/lpr mice. Several lines of evidence support this. First, our previous findings showed that the MRL/MpJ mice heal similarly to MRL/MpJ-Fas^(lpr) mice (36). Secondly, intervals containing wound-healing genes in our crosses showed no overlap with those from another report on the genetic analysis of MRL/lpr autoimmune phenotypes (17). Third, we tested the lymph node cell number, a parameter associated with lymphoproliferation, of each of 101 F₂ mice in a regression comparison with their healing phenotype, and no association was found with the healing trait (r²=0.0002, p=0.89) (Blankenhorn, et al., in preparation).

The MRL/lpr mouse strain was originally selected for its large size (13, 37); it was subsequently found to have a major defect in immune regulation, due to a retrotransposon insertion into the second intron of the fas gene (16, 54). This mouse exhibits immunological defects closely mimicking those of the human disease, systemic lupus erythematosus (SLE) and other lymphoproliferative disorders (14, 15). In the present study, however, none of the five healing QTLs nor the highly suggestive regions identified displays linkage to the fas gene or other genes proposed thus far to control the other autoimnmune phenotypes seen in this strain of mouse.

EXAMPLE 10

Optic Nerve Regeneration in MRL Mice.

Healer (MRL/+) and non-healer (C57Bl/6) mice were anesthetized and the optic nerve was cut using a micro-dissecting spring scissors from behind the left eye socket. The optic nerve was visually inspected to identify a cut end. The right eye was not touched. The mice were examined after 1.5 months.

Tissue from both the control right eye and the left eye where the-optic nerve had been transected was removed, fixed in formalin, and embedded in paraffin. Serial sections were made and stained with hematoxylin and eosin. Several tissue sections were destained and restained with Bodian's silver stain to visualize axons.

In the all three non-healer mice the left eye had completely atrophied and had sunken into the eye socket while the control, right eye appeared normal (FIG. 9B). There was no evidence of eye-specific tissue in the left socket, although lacrimal gland tissue was present. The optic nerve near the eye and the chiasm had disappeared. Thus, there was no evidence of the presence of any part of the left eye or the associated optic nerve remaining in these animals.

In contrast, in all four healer mice the left eye had either only slightly sunken or was of normal size when compared to the right (control, non-treated) eye which was normal (FIG. 9A). The optic nerve could be found in both eyes of all four healer mice and the chiasm appeared normal. The right (control, non-treated) optic nerve appeared normal in each case.

Histologic sections of eye-specific tissue from both healer and non-healer mice were examined 1.5 months after the optic nerve of each mouse's left eye had been surgically transected. In the healer mice, longitudinal serial sections of the right (control) eye tissue exhibited a normal, uncut optic nerve (FIG. 10). However, histologic sections of tissue from the left eye of healer mice demonstrated, in the first and last sections, a cut optic nerve (FIG. 11). More importantly, the middle histologic sections exhibited an increasing neural connection between the cut regions of the optic nerve. FIG. 11 shows an example of a restored optic nerve region as being thinner and irregular when compared with the uncut optic nerve of the (control, non-treated) right eye shown in FIG. 10.

Further, there appeared to be connective tissue and infiltrating macrophages filled with melanin as well as a proliferation of glial cells, especially oligodendrocytes, in the left eye-specific tissue of healer mice. Additionally, a blood vessel can be seen running along side the restored optic nerve in FIG. 11. This observation is important because cross-sections of uncut normal and healer optic nerves have blood vessels which run along side and are embedded into the optic nerve. These blood vessels would have been unavoidably cut upon transection of the optic nerve. Thus, it appears that the healer mice demonstrated regrowth of the blood vessels running along the restored optic nerve in addition to regeneration of the optic nerve itself.

Several sections were then destained and restained to visualize axons with Bodian's silver stain. As can be seen in FIG. 12A, the healer uncut (right) eye has bundles of nerves woven through the optic nerve. On the other hand, the cut (left) healer optic nerve (shown in FIG. 12B) has a left optic nerve which appears to be generally depleted of nerve fibers. On the side of the healed optic nerve nearest to the eye, nerve fibers can be seen growing towards the brain. These fibers can be seen leaving a nerve cell body and having directionality (FIG. 12B). Near the newly grown nerve fibers are an abundance of oligodendrocytes.

Thus, healer MRL mice can regenerate nerve tissue. More specifically, all four MRL mice in which the left optic nerve was transected exhibited regrowth of the optic nerve within 1.5 months after transection. In these mice, the left optic nerve has clearly been cut but a neural connection was reestablished, with marked cellular proliferation including infiltrating macrophages filled with melanin and oligodendrocytes. Further, vascular connections had also been reestablished in the left healer eye as demonstrated by the presence of a blood vessel running along the side of the restored optic nerve. By comparison, the non-healer mice had no eye-specific tissue in the left eye socket and no nerve regeneration was detected.

Nerves which have been examined using the above-described procedures are the peripheral nerve, including the sciatic nerve, and central nervous system nerves including the optic nerve, brain stem and spinal cord.

EXAMPLE 11

Restoration of Function in MRL Mice Following Sciatic Nerve Crush.

Three strains of mice, C57BL/6, MRL and A/J, were examined after sciatic nerve crush. The percent of sensory function index (SFI), which is related to sciatic nerve function, is the comparison of the gait of the mouse having a normal left and a cut right sciatic nerve. To measure the SFI, the footpads of the animals were inked, and the animals were allowed to walk on a paper pad in a housing which forces them to walk forward. The pattern of ink deposition is directly related to theSFI, which was then calculated for each mouse.

The results of these experiments are shown in FIG. 13. Significant recovery of sciatic nerve function was observed in MRL mice (50% of ⅔ mice) at day 30. In contrast, C57BL/6 and A/J mice exhibited an average negative SFI value, indicating that the sciatic nerves in these animals did not recover.

EXAMPLE 12

Involvement of T Cells in Healing in Aged Healer Mice.

Several facts indicated that T cells might play a role in wound healing in aged healer mice. First, scarless healing occurs in fetal mice until embryonic day 16, when T cells develop in the thymus. Second, in MRL/lpr mice, the number of lymphocytes increases with age. Third, aged healer mice do not heal as well as younger healer mice (FIG. 17). We therefore examined the role of T cells in the enhanced healing response of these mice in two ways.

First, we treated aged (5 month-old) healer mice with antibodies against T cell receptors and punched a 2 mm ear hole in the treated aged mice. As measured by this assay, aged mice which were treated with anti-TCR antibodies became complete healers, similar to their younger counterparts (FIG. 18).

Second, we crossed a member of the F₁ generation from a cross between a healer and a non-healer mouse whose T cell receptors had been genetically eliminated, or “knocked out.” Members of the resulting F₂ generation were then examined for a healer phenotype using an ear punch assay and for the presence of T cells by flow cytometry. Approximately 12% of the F₂ generation had no T cells and were complete healers. (FIG. 19).

These results suggest that T cells somehow suppress the healing response in aged healer mice and that elimination of functional T cells restored the healing capacity of the mice. T cells obtained from non-healer mice can now be transferred into healer mice to determine whether the T cells, or a particular population of the T cells, have an effect on the enhanced wound healing of healer mice.

EXAMPLE 13

Adoptive Transfer of Fetal Liver Cells from Healer Mice into X-Irradiated Non-Healer Recipients Enhances Wound Healing.

C57BL/6 mice were lethally irradiated. After 24 hours, each mouse received an injection of fetal MRL liver cells into the tail vein. Ear punches were performed at 2, 4, and 6 months after injection of the liver cells.

No healing was observed in the ears of mice after 2 or 4 months. After 6 months, however, ear holes of the C57BL/6 (non-healer) mice exhibited a healing response. This response was correlated with the detection of chimeric cells in the thymus and/or lymph node of the mice, as shown in FIG. 20.

Thus, it is possible to transfer the ability to heal ear holes to adult non-healer mice using adoptive transfer of fetal liver cells from a healer mouse strain.

EXAMPLE 14

Adoptive Transfer of Macrophages Obtained from Healer Mice to Non-Healer Mice Recipients Enhances Wound Healing.

The MRL mouse is derived from crossbreeding of LG (75%), AKR (12%), C3H (12%) and C57BL (0.3%) mice. Wild type strains of these mice were tested for their ability to heal or to partially heal 2 mm ear hole wounds. The only mouse which exhibited partial healing was the LG mouse (FIG. 14). The experiment was conducted as described herein for the MRL mouse.

Normal C57BL/6 mice were injected intraperitoneally with macrophages obtained from healer mice at the time of ear punching of the non-healer mice. The macrophages which were transferred were five day thioglycollate induced peritoneal cells obtained from an (MRLxB6)F₂ T cell receptor alpha chain knockout mouse. This mouse was used because the αβ+T cells were absent and the cells were only partially allogeneic.

Macrophages were obtained from healer mice using standard procedures, selected by adherence to plastic, and transferred to non-healer mice. Uninjected non-healer mice and non-healer mice injected with non-healer macrophages served as controls in the experiment. Ear hole closure was measured during the 30 days after wounding. The results of the experiment are shown in FIG. 15.

Healer macrophages induced the best ear closure (greater than 75%) in non-healer mice. (FIG. 21). Macrophages which were obtained from non-healer mice and which were transferred into non-healer mice were unable to confer healing on non-healer mice. These results establish that tissues and cells obtained from healer mice can be used to confer healing ability on non-healer mice.

EXAMPLE 15

Functional Recovery of Healer Mice Following Complete Transection of the Spinal Cord.

A complete transection of the spinal cord at thoracic level (TH9-TH11) was made in MRL and in C57BL/6 mice anesthetized with ketamine/xylazine (100/15 mg/kg, i.p.). A dorsal median incision was made over the lower thoracic vertebra. Retractors were placed in the wound to allow surgical manipulation. After exposing the vertebral arch and the spinous processes by retracting the medial part of the autochtone musculature, the osseous parts of one vertebral bone were removed with a milling cutter, leaving the dura mater intact. After continuous cooling of the spinal cord with ice cold isotonic saline to minimize bleeding, transection was performed with a scalpel. In some mice, a portion of the spinal cord was removed, rather than simply cut. In these mice, more of the spinal cord is exposed, by removing only the caudal part of the more cranial vertebral arch. The cranial part of the more caudal vertebral arch was also be removed to minimize vertebral instability. Two transections were performed, and the tissue between the transections was removed with a fine forceps.

Ice cold isotonic saline was then applied in pulses until no bleeding occurred. The muscles of both sides were repositioned and gently opposed by single 8-0 resorbable sutures. The skin was closed in the usual manner with single sutures using 8-0 Dexon (Polyglycolic Acid).

Animals were weighed daily to ensure proper water and nutritional intake. Food and water was made available at ground level through a special device. In the first postoperative days, each mouse was watched carefully for its ability to eat and drink, unless it was fed manually. The bladder was emptied 3 times a day by applying gentle pressure to the abdomen. The mice were also carefully watched for potential injury and self mutilation due to loss of innervation of the hind limbs.

After 2 days of postoperative recovery, the mice were examined for loss of lower limb innervation. Both strains showed no innervation of the hind limbs, which were dragged behind. After 2 weeks, neither strain of mice showed signs of innervation of the hind limbs. Between seven and nine weeks, however, MRL mice regained partial proximal innervation of the tail and lower limbs, exhibiting the ability to flex and then extend their hind legs. C57BL/6 mice continued to show no movement of the hind limbs or tail and to show degeneration of the limb musculature and fixed flexion of the joints. In contrast, fixed flexion of the joints of the MRL mice never occurred, and only minor muscle degeneration was in the observed first month.

Histological examination revealed that MRL mice produced only aminor scar between the two ends of the transected spinal cord (2-5 cell layers), whereas C57BL/6 mice produced a conventional glial scar (10-15 cell layers), thereby preventing the spinal cord to reconnect on an axonal level.

The partial functional recovery in MRL mice, as well as the underlying histomorphology, suggests that regeneration and reconnection on the axonal level of the spinal cord of MRL mice can occur after complete transection of the spinal cord.

EXAMPLE 16

Differential Expression of Genes in Dendritic Cells of Healer and Non-Healer Mice.

Dendritic cells (immature macrophages) were isolated from the bone marrow of adult C57BL/6 and MRL⁺ mice using standard procedures. Total RNA was isolated from the dendritic cells and subjected to microarray analysis, as described above in the Materials and Methods section.

Table 15 shows genes whose expression was increased more than 50% in either MRL⁺ or C57BL/6 mice. The gene names and array locations are from the Clontech web site, available at the URL address: http file type, www host server, domain name clontech.com, chlontechIJAN98UPD/Atlaslist directory.

EXAMPLE 17

An In Vitro Assay of Wound Healing.

A hole was punched in the ear of a healer and a non-healer mouse. Two days later, an explant of the ear tissue, including the punched hole, was excised, embedded in a collagen gel, and maintained in vitro.

FIG. 22 shows explants of healer (FIGS. 22A, B) and non-healer (FIGS. 22C, D) ear punch explants in vitro. FIG. 23 shows cells migrating away from the explant of the healer ear punch, possibly in response to factors which are produced by the wounded ear.

EXAMPLE 18

Adoptive Transfer of Bone Marrow-Derived Dendritic Cells from Healer Mice into Non-Healer Immunodeficient Mice Enhances Healing.

Dendritic cells were isolated from healer (MRL⁺) and non-healer (C57BL/6) mice as described in Example 16. C57BL/6 Rag^(−/−) mice, which are immunodeficient and unable to reject the dendritic cells, were ear-punched to make a 2 mm hole and immediately injected intraperitoneally with dendritic cells of either MRL⁺ or C57BL/6 mice. Ear hole closure was measured at 0, 2, 5, 10, 18, 24, and 27 days after wounding. The results of this experiment are shown in FIG. 23.

Healer dendritic cells induced the best ear closure (approximately 90%) in the non-healer mice, demonstrating that the healer dendritic cells can be used to transfer enhanced healing ability to non-healer mice.

EXAMPLE 19

Segregation of Quantitative Trait Loci in Phenotype Congenic Mice.

Congenic mice were generated by six successive back-crossings of the F₁ generation of a healer (MRL)×non-healer (C57BL/6) cross with its non-healer parent strain. Segregation of quantitative trait loci was examined in healer phenotype congenic mice (see Examples 5-8, above). Each of the 17 phenotype congenic mice examined healed a 2 mm ear hole from at least 75% to 100% 30 days after punching.

Table 16 shows that only certain markers segregate with the healer phenotype in these phenotype congenic mice. These mice are therefore useful for dissecting out the minimum number of chromosomal loci and genes involved in enhanced wound healing.

TABLE 1 Residual wound size Backcross Parental strains and hybrid and intercross progeny C57BL/6 1.10 ± 0.27 F1 × MRL/lpr 0.35 ± 0.27 MRL/lpr 0.04 ± 0.06 F1 × B6 0.95 ± 0.25 (MRL × B6)F1 0.73 ± 0.22 (MRL × B6)F2 0.83 ± 0.28 Numbers are the diameter of holes in mm ± standard deviation (SD) on day 28-30. F1 = (MRL × C57BL/6)F1 hybrid.

TABLE 2 Location of heal QTL as determined in the intercross (F2) and the backcross (BC1) progeny. MGD F2 F2 BC1 BC1 cM Primers LRS¹— p value LRS²— p value QTL 71.5 D1Mit288 6.1 0.0130 ns 105 D2Mit148 nd 4.8 0.0279 55.6 D4Nds2 4.2 4.2 0.0400 ns 77.5 D4Mit127 5.5 0.0190 ns 52.4 D7Mit220 ns 10.2* 0.0014 21 D8Mit191 ns 6.4 0.0115 33 D8Mit132 5.5 0.0188 ns 37 D8Mit249 7.3 0.0069 ns 49 D8Mit211 10.7  0.0011 ns heal1* 56 D8Mit166 7.3 0.0068 nd 34 D12Mit4 ns 7.1 0.0077 52 D12Mit233 6.1 0.0137 9.4 0.0022 52 D12Mit132 nd 10.9  0.0009 heal5*^(#) 9 D13Mit135 9.4 0.0022 4.8 0.0290 11 D13Mit115 9.7 0.0019 5.0 0.0261 heal2*^(#) 13 D13Mit116 8.7 0.0030 5.1 0.0230 30 D13Mit245 7.5 0.0335 nd 32 D13Nds1 10.2* 0.0014 ns 44 D13Mit126 7.4 0.0065 ns 44 D13Mit191 10.2* 0.0014 ns 47 D13Mit29 6.5 0.0101 nd 48 D13Mit107 7.4 0.0064 ns 49 D13Mit144 6.9 0.0088 nd 60 D13Mit129 10.8  0.0010 8.3 0.0040 heal3*^(#) 62 D13Mit53 10.5  0.0012 7.1 0.0077 71 D13Mit151 8.2 0.0042 4.6 0.0318 54.5 D15Mit171 6.6 0.0104 nd 55.6 D15Mit242 7.1 0.0079 nd 57.8 D15Mit172 7.3 0.0070 nd 56.8 D15Mit244 10.7  0.0011 nd heal4* 56.8 D15Mit14 8.5 0.0035 ns Underlining indicates the LRS values that were used for assigning heal QTL. Footnotes: ¹Threshold LRS for significant linkage = 10.7, and for suggestive linkage, 3.3 ²Threshold LRS for significant linkage = 11.8, and for suggestive linkage, 3.7 *see text for discussion ^(#)confirmed in second cross

TABLE 3 Single locus genotypic values for heal1 to heal5. Genotype avg stdev p* avg stdev p D8Mit211 (heal1) D13Mit115 (heal2) b/b 0.73 0.27 0.0319 0.96 0.25 0.0904 b/s 0.84 0.27 0.1214 0.84 0.29 0.0567 s/s 0.95 0.28 0.0013*** 0.72 0.26 0.0024*** D13Mit129 (heal3) D15Mit244 (heal4) b/b 0.96 0.28 0.2156 0.95 0.19 0.0485 b/s 0.84 0.22 0.0141** 0.86 0.30 0.0677 s/s 0.74 0.34 0.0022*** 0.70 0.27 0.0013*** D12Mit132 (heal5) b/s 0.51 0.40 0.0005*** s/s 0.15 0.32 Averages and standard deviations for wound diameters at day 30 are given for mice sorted by the genotype of markers near each healing QTL. *, p values in the F2 are listed vertically for b/b vs. b/s, s/s vs. b/s, or b/b vs. s/s; in the backcross, p values are for the heterozygote b/s vs. the homozygote s/s. Genotype values are significant at α = 0.05 (**) or α = 0.01 (***) in post-hoc analyses.

TABLE 4 Candidate genes in genomic intervals containing QTLs QTL MGD cM Candidate genes in interval heal1 33 Comp, cartilage oligomeric matrix protein 39 pdw, proportional dwarf 42 Os, oligosyndactylism 46 Gna0 51.5 to 67 Cadherin family heal2 7 Nid, nidogen 8 Gli3, GLI-Kruppel family member GLI3 10 Amph, amphiphysin 10 Inhba, inhibin beta-A 10 Rasl1, Ras-like, family 1 heal3 32 Msx2, hox8 32.5 Fgfr4, fibroblast growth factor receptor 33 mes, mesenchymal dysplasia 36 Tgfbi, transforming growth factor induced 44 Cspg2, chondroitin sulfate proteoglycan 45 Rasa, ras p21 GTPase activating protein 56 Gpcr18, G-protein coupled receptor 18 62 Itga 1,2, integrin alpha 2 (Cd49b) heal4 51.6 Pdgfec, platelet derived growth factor 56.8 Col2a1, procollagen, type II, alpha 1 56.8 Ela1, elastase 1 57 Emb, embigin 57.1 Hoxc, homeo box C cluster 57.1 Rarg, retinoic acid receptor, gamma 57.5 Dhh, desert hedgehog homolog 58.7 Krt2, keratin gene complex 2 60 Itga5, integrin alpha 5 61.1 Itgb7, integrin beta 7 63 Glycam1 adhesion molecule heal5 40 Fos, FBJ osteosarcoma oncogene 41 Tgfb3, transforming growth factor, beta 44.6 Chx10, C elegans ceh-10 homeo domain con 45 Pgf, placental growth factor

TABLE 5 Differentially expressed or regulated genes identified using microarray analysis of mRNA obtained from healing tissue on days 5 and 20 after ear punch compared to tissue obtained on day 0. Gene Group* Chromosome # Gene IA 1 Stat 1 1 PAI-2 2 ABI-2 2 Integrin alpha 2 Basic domain transcription factor 2 SEF-2 4 TNFR-1 4 SKI 7 H-ras 7 Cathepsin D 8 Glutathione Reductase 10 TIMP-3 10 STAT6 11 ACE 11 Macrophage inflammatory Protein 11 c-erb 14 BMP-1 14 Cathepsin B 16 HMG 16 ETS 18 c-FMS IB 2 CRAPBII (RA binding protein) 2 CD44 2 SKY proto-oncogene 8 Casein kinase 12 YY1 12 c-AKT 13 GTT 13 SPI-3 13 GKLF 13 Cathepsin L 13 PDGF signalling molecule 13 thrombin receptor 15 epidermal keratin *Genes in Group II are listed in Tables 10 and 11. Table 10 is a list of genes which are differentially expressed on days 5 and 20 after ear punch between control mice (B6 & F₂ nonhealers) and experimental mice (MRL & F₂ healers). Table 11 is a list of genes which are regulated in MRL tissue from 0, 24, and 40 hrs after wounding or ear punching (see FIG. 16).

TABLE 6 Differentially expressed or regulated genes identified using RT-PCR analysis of mRNA from healing tissue. Gene Group Chromosome # Gene IB 13 MSX-2 15 RAR-gamma

TABLE 7 Differentially expressed genes in Groups I and II identified using differential display analysis of mRNA from healing tissue.* Gene Group Chromosome # Gene IB 8 ecadherin II PMG-1 TR 2-11 tropomyosin *RNA from healer and nonhealer ears on days 0, 5 and 20 after ear punching were analyzed by RT-PCR. Random primers obtained from “Gene Hunter Corp” were used to amplify bands. Since these are random primers, the products are unknown but can be sequenced. It is a useful way of examining what amplifies in one population and not another. Thus, one has to compare what is present or not in B6 and MRL for each time point and then subtract out the differences seen in day 0.

TABLE 8 Differentially expressed genes from Groups I and II identified using SAGE analysis of mRNA from healing tissue. Gene Group Chromosome # Gene IB 15 epidermal keratin II macrophage ferritin heavy subunit est (1758377) and see Table 13

TABLE 9 Microsatellite Markers Linked to Male and Female Healer Mice CHROM # MARKER LOCATION(CM LRS CROSS  1 D1MIT64 21 5.1 F2-Male  1 D1MIT123 36.9 4 F2-Male  1 D1MIT288 71.5 7.5 F2  1 D1MIT356 95.8 4.3 F2-Female  2 D2MIT329 45 5.6 F2-Female  2 D2MIT37 45 6.3 F2-Female  2 D2MIT14 49 8 F2-Female ##2  D2MIT207 60 10.5 F2-Male; F2  2 D2MIT107 75.6 7.9 F2-Male  2 D2MIT223 76.7 9.3 F2-Male  2 D2MIT148 105 4.8 Backcross  3 D3MIT60 0 6.3 F2  3 D3MIT203 11.2 4.3 F2  3 D3MIT310 38.3 5.4 F2; F2-Male ##4  D4MIT149 0 13.4 F2-Male  4 D4MIT235 2 9.8 F2-Male  4 D4MIT236 12 5 F2-Male  4 D4MIT241 25 5.5 F2-Male  4 D4MIT127 78 8.8 F2-Male  5 D5MIT148 18 5.8 F2-Female  6 NONE ##7  D7MIT220 52 10.2 Backcross  7 D7MIT237 66 6.6 Backcross  8 D8MIT191 21 6.4 Backcross #8 D8MIT211 49 9.9 B6  9 NONE 10 D10MIT42 44 3.5 F2-Female 10 D10MIT233 62 4.2 F2-Male 11 D11MIT124 61 8.1 F2-Male; F2 12 D12MIT4 34 7.1 Backcross 12 D12MIT233 52 9.4 Backcross ##12  D12MIT132 52 10.9 Backcross 13 D13MIT135 10 9.4 F2 13 D13MIT115 11 9.7 F2 13 D13MIT116 13 9 F2 13 D13MIT245 30 8.8 F2 ##13  D13NDS1 32 15.9 F2 13 D13MIT126 45 6.6 F2 13 D13MIT191 45 9.4 F2 ##13  D13MIT159 45 12.7 F2 13 D13MIT29 47 6.5 F2 13 D13MIT107 48 7 F2 13 D13MIT144 48 6 F2 ##13  D13MIT148 59 11.9 F2 ##13  D13MIT129 60 11.2 F2 13 D13MIT53 62 9.1 F2 13 D13MIT151 71 8.2 F2 14 D14MIT193 40 5 F2-Male 14 D14MIT131 58 4 F2-Male 15 D15MIT189 48.5 6.6 F2; F2-Female 15 D15MIT171 54.5 6.7 ″ 15 D15MIT242 55.6 6.7 ″ 15 D15MIT172 57.8 6.9 ″ ##15  D15MIT242 55.6 10.7 ″ 15 D15MIT14 56.8 9.3 ″ 15 D15MIT43 60.4 4.6 ″ 15 D15MIT79 66.2 7.9 ″ 15 D15MIT16 61.7 5.9 ″ 16 D16MIT122 3.85 6.2 F2 16 D16MIT32 48.2 6.6 F2 17 D17MIT68 24.5 5.7 F2 18 D18MIT123 31 4.8 F2 18 D18MIT49 49 4.1 F2 19 D19MIT59 0.5 7.2 F2-Female The suggestive loci have an LRS value of 3.5 or greater The significant loci have an LRS value of 10.5 or greater (except when confirmed in a second cross (##)

TABLE 10 Wound Healing Gene Products Identified by Microarray Analysis Gene Products Identified by Day Chrom. microarray analysis Day 5 20 Local. (day 5 and 20 healer and nonhealer Express. Exp. (cM) ear holes compared to day 0) up up ? Ezrin; Villin 2; NF-2 (merlin) related filament/plasma membrane associated protein no change DOWN 14 (41) Rb; pp105; Retinoblastoma susceptibility-associated protein (tumor suppressor gene; cell cycle regulator) up (female) 7 TSG101 tumor susceptibility (syntenic) protein down down ? Tumor suppressor maspin (female) (female) down (male) down 7 (28.5) ZO-1; Tight junction protein; (male) discs-large family member, partially homologous to a dlg-A tumor suppressor in Drosophila down (male) down 11 (57) c-ErbA oncogene; thyroid hormone (male) receptor up up 4 (44.6) c-Jun proto-oncogene (transcription factor AP-1 component) no change down ? RNA polymerase I termination factor TTF-1 up up 15 (32) c-myo proto-oncogene protein down up 8 (49.5) Casein kinase II (alpha subunit) (female) (female) down (male) down 17 (16.4) Pim-1 proto-oncogene (male) up (female) 18 (30) c-Fms proto-oncogene (macrophage colony stimulating factor 1 (CSF-1) receptor) up (female) up 5 (42) PDGFRa; platelet-derived growth (female) factor alpha-receptor down (male) down 4 (78.9) Ski proto-oncogene (male) up 2 (67) Sky proto-oncogene (Tyro3; Rse; Dtk) up (female) 7 (72.2) H-ras proto-oncogene; transforming G-protein down 3 (48.5) N-ras proto-oncogene; (male) transforming G-protein up (male) 1 (36.1) IGFBP-2; Insulin-like growth factor binding protein 2; autocrine and/or paracrine up (female) ? Cyclin B2 (G2/M-specific) down up ? Cyclin D2 (G1/S-specific) up (male) down ? Cyclin G (G2/M-specific) (male) up down ? p18lnk4; cdk4 and cdk6 Inhibitor down up ? Prothymosin alpha up ? HSP84; heat shock 84 kD protein up ? HSP86; heat shock 86 kD protein up 2 (22.5) Glucose regulated protein 78 kD; Grp78 up (male) 3 (66.2) Golgi 4-transmembrane spanning transporter; MTP up 13 (47) C12r; coagulation factor II (thrombin) receptor up (female) ? Interleukin-6 receptor beta chain; membrane glycoprotein gp130 up 19 (0.5) 1-kappa B alpha chain up (male) down 1 (28) Stat1; signal transducer and (male) activator of transcription up (male) down 10 (70) Stat6; signal transducer and (male) activator of transcription 6; IL-4 Stat; STA6 up 11 Crk adaptor protein (syntenic) up down 17 (40) Inhibitor of the RNA-activated protein kinase, 58-kDa up ? MAPK; MAP kinase; p38 UP DOWN 9 (36) MAPKK1; MAP kinase kinase 3 (dual specificity) (MKK1) up down 2 (2) PKC-theta down down 13 (50) Pl-K p58, PDGF signalling pathway member up (male) down ? Rab-2 ras-related protein (male) up (male) down ? 14-3-3 protein eta (male) up 11 (29) IRF1; Interferon regulatory factor 1 UP DOWN ? Zyxin; LIM domain protein; alpha- actinin binding protein up (male) down ? BAG-1; bcl-2 binding protein with (male) anti-cell death activity up up 13 (8) Glutathione peroxidase (plasma protein); selenoprotein up 8 (16) Glutathione reductase DOWN DOWN ? Glutathione S-transferase (microsomal) down (male) ? Glutathione S-transferase Mu 1 up down 12 (58) c-Aki proto-oncogene; Rac-alpha; proteine kinase B (PKB) up down ? FLIP-L; apoptosis inhibitor; FLICE-like inhibitory protein up (female) down 3 (70.5) Gadd45; growth arrest and DNA- (female) damage-inducible protein up ? Nm23-M2; nucleoside diphosphate kinase B; metastasis-reducing protein; c-myc-related transcription factor up (male) down ? Protein tyrosine phosphatase (male) up (male) down 19 (23) RIP cell death protein; Fas/APO-1 (female) (CD95) Interactor, contains death domain UP DOWN 13 (16) SPl3: serpin; similar to human proteinase inhibitor 6 (placental thrombin inhibitor) serine proteinase inhibitor UP DOWN 4 (75.5) Tumor necrosis factor receptor 1; TNFR-1 up (female) UP ? PA6 stromal protein; RAG1 gene (female) activator up ? MHR23B; Rad23 UV excision repair protein homologue; xeroderma pigmentosum group C (XPC) repair complementing protein up 2 (45) Abiphilln-1 (abl-1) similar to HOXD3 UP x-14 Adipocyte differentiation- associated protein H >> NH H >> NH 2 (91) Basic domain/leucine zipper transcription factor up ? Butyrate response factor 1 NH >> H NH >> H ? CACCC Box-binding protein BKLF up ? DP-1 (DRTF-polipeptide 1) cell cycle regulatory transcription factor down up 16 (68.5) Els-2 transcription factor (female) (female) down up 2 (7) GATA-3 transcription factor (female) (female) down up 13 (16) Gut-specific Kruppel-like factor (female) (female) GKLF down up 16 (69) HMG-14 non histone chromosomal (female) (female) protein up NH > H ? Interferon regulatory factor 2 (IRF 2) up NH > H ? NF-1B protein (transcription factor) up down 15 (61.7) Nuclear factor related to P45 NF-E2 up (male) down 9 (36) Nuclear hormone receptor (male) ROR-ALPHA-1 up down 6 Split hand/foot gene (syntenic) down up 2 (54) Retinoic acid binding protein II (female) (female) cellular (CRABP-II) down up ? Retinoid X receptor Interacting (female) (female) protein (RIP 15) up down ? Transcription factor 1 for heal shock gene up ? Transcription factor CTCF (11 zinc fingers) NH >> H up 2 (94) Transcription factor SEF2 (female) down up ? YB1 DNA binding protein (female) (female) up (female) up 12 (53) YY1 (UCRBP) transcriptional (female) factor NH >> H up ? Activin type I receptor down (f) (female) up (male) down ? C—C chemokine receptor (male) (Monocyte chemoattractant protein 1 receptor (MCP-1RA) down down ? Growth factor receptor (female) (female) NH >>> H up 18 (20) Glucocorticoid receptor form A up up 6 (31.5) CD31 (Platelet, endothelial cell adhesion molecule 1) up up 2 (56) CD44 antigen up 9 (56) Dystroglycan 1 up down ? Glutamate receptor channel subunit gamma up 2 (38) Integrin alpha 6 UP (female) up ? Integrin beta (female) UP (female) up ? Laminin receptor 1 (female) up (male) UP 14 (32.5) Bone morphogenetic protein 1 (female) NH >> H up ? Insulin-like growth factor binding protein-6 (IGFBP 6) up 11 (2) Insulin-like growth factor binding protein-3 (IGFBP-3) UP ? Insulin-like growth factor binding protein-4 (IGFBP-4) UP 1 (36) Insulin-like growth factor binding protein-5 (IGFBP-5) UP (female) UP 10 (48) Insulin-like growth factor-IA (female) up 11 (47.6) Macrophage inflammatory protein down 18 (48) Mad related protein 2 (MADR2) (male) up 7 (11) Neuroleukin UP 14 (20) Placental ribonuclease inhibitor (female) (Anglogenin) up down 7 (6.5) Transforming growth factor beta up down 7 (6.5) Transforming growth factor beta 2 up (female) x (1.7) Cytoskeletal epidermal keratin (14 human) H >>> NH UP 15 (58.7) Epidermal keratin (1 human) UP up (male) down 9 (61) Non-muscle myosin light chain 3 (male) up (female) 2 (7) Vimentin up down 11 (65) Angiotensin-converting enzyme (ACE) (clone ACE.5.) up 14 (28.5) Cathepsin B down 7 (72.5) Cathepsin D up 13 (30) Cathepsin L H >> NH down ? Cytotoxic T lymphocyte-specific up (f) (male) serine protease CCP I gene (CTLA-1) NH >> H UP 14 (20) Mast cell protease (MMCP)-4 UP ? Membrane type matrix (female) metalloproteinase DOWN 1 (61) Plasminogen activator inhibitor-2 UP ? Serine protease Inhibitor homolog J6 NH >>> H 10 (47) TIMP-3 tissue inhibitor of up metalloproteinases-3

TABLE 11 Genes whose expression is altered in MRL mice 0 hrs., 24 hrs., and 40 hrs. after ear punch. Block A Block B Block C Block D Block E Block F 1a. APC 1a. HSP-27 1a. Caspase -11 1a. abi-1 1c. BMP-R 1b. BMP-1 1b. BRCA1 1b. HSP-60 1e. bag-1 1j. BKLF 1g. CSA-R 1d. BMP-4 1e. EB1 1c. HSP-84 1f. Bak 1k. C/EBP 2b. FGFR-4 1g. Cek 5 1f. ezrin 1d. HSP-86 1g. Bax 2g. DP-1 2e. GMCSF-R 1h. Cek 7 1i. NF2 1f. Osp-94 2a. GTT trnfs. 2j. Elf-1 2l. INFab-R 1j. EGF 1j. p107 1j. cyp1b1 2b. GTT Mu-1 3m. HMG-14 2m. INFg-R 2a. G-CSF 1l. p53 2e. Glut1 2d. GST pi-1 3n. Hox1.1 3a. IL-1R 2i. IGFBP-6 2a. maspin 3d. CXCR4 2e. A20 znc 4g. Hox 8 3c. IL2-R 2k. IGFBP-3 fnger pr. 2d. VHL 3f. Prostaglandin E R 2k. c-Akt 4m. LKLF 3d. IL3R 2l. IGFBP-4 2g. c-erb 3g. Tie-1 3a. CHOP 10 4n. Lbx1 3g. IL-7R 2m. IGFBP-5 2j. TTF-1 3j. Vegfr-2 3b. Clusterin 5a. Mph-1 3h. IL8-R 3a. ILGF-14 2l. c-myc 4h. TANK 3f. Fas1 R 5b. MRE 3l. Estrog R 3c. K-FGF 3b. FLI-1 4k. Tristetraproline 4c. Nm23-M2 5h. NF-E2 3j. Androg R 3g. MIP2 3e. Gli 5b. Hck 4g. PtPhosphatase 5m. DSS-1 3m. Glucocort R 3h. MADR2 3l. B-Raf 5f. CamK 4h. PS-2 5n. Sox3 4a. Insulin R 3j. Smad 1 4e. PDGFRa 5h. Erk1 5a. TDAG51 6b. PAX-6 4b. IRS-1 3k. NGF (fas reltd) 4g. SKI 5j. Jak3 5b. TNF 55 6f. RXR-6 4h. Serotonin R 1e−b 3m. neuroleukin 4j. Vegfr-1 6h. PKC-theta 5d. TNFR-1 6g. RIP-15 4j. Serotonin R 3 3n. oncostatin 4n. c-Src 6k. PI3-K 6j. MHR23B 6i. TF-1 for HSP 4m. Adrenergic R beta 1 4d. thrombomod. 5c. H-ras 7b. Rab-2 7b. PCNA 7e. SEF2 5c. GPCR 4g. TGFb2 5d. LFC 5f. GABA-A 3 5b. IL-4 5j. b-protachykinin 7j. GapIII 7c. bluelight R 7g. SP2 5g. GABA-A 4 5c. IL-6 5m. IGFBP-2 5l. P-selectin 5d. IL-7 6a. cyclin A 7n. Zyxin 7e. Pur 5m. catenin alpha 5e. cardiac myosin 6d. cyclin B2 6a. CD2 5g. CDC42 bind. prot. 6h. cyclin D3 7k. HR6B 6h. CD14 5h. epidermal keratin-14 7b. p58/GTA 6m. dystroglycan 5i. epid.k-18 7e. cdk inhibit prot. 7n. XRCC1 6n. glutamate R 5k. epiderm keratin 7j. cdc25a 7a. CD49b 5n. kinesin 7m. prothymosin a 7c. CD51 6b. nonmuscle myos. 7g. Integrin beta 6d. vimentin 7i. ICAM-1 6e. unconv myosin VI 7j. Laminin R 1 6g. cathep B 7l. Neuronal prot F3 6h. cathep D 7n. VLA-3 6i. cathep H 6j. cathep L 6k. collagenase IV 6m. CTLA-1 6n. gelatinase B 7b. MMCP-4 7c. MTMMP 7e. TPA 7g. Protease Inhib 2 7i. PAI-2 7j. Spi-2 7n. TIMP-3

TABLE 12 SAGE Analysis of C57BL/6 Mice Five Days After Wounding SearchName = B5BANK FileName c:\sage\data\b5data-1\tagbank.bag Anchoring Enzyme NlaIII-CATG Tag Length = 11 DiTag Length = 24 Total Files = 56 Total Tags = 1508 Total Duplicate Dimers = 0 Tag Abundance Report Total tags after excluding tags = 1508 Count Percent Tag Sequence −Tag BaseFour Number SEQ ID NO: 16 1.601 GGCTTCGGTCT −2750136 1 13 .862 AGAGCGAAGTG −563247 2 12 .7957 AGCAGTCCCCT −601432 3 12 .7957 GTGGCTCACAA −3054865 4 10 .6631 AGGCAGACAGT −673868 5 9 .5968 GCTGGCCCTTC −2598270 6 8 .5305 AGGTCGGGTGG −711355 7 8 .5305 CGCCGCCGGCT −1664424 8 7 .4641 CTGCTCAGGCT −1995944 9 7 .4641 TGGGCATCCAC −3838802 10 6 .3978 AAGGAAATGGG −164075 11 6 .3978 ATACTGACATT −817232 12 6 .3978 CCTTTGTGACT −1571720 13 6 .3978 GAACATTGCAC −2117522 14 6 .3978 GGGAAGGCGGC −2755178 15 5 .3315 ATGACTGATAG −925235 16 5 .3315 CACAAACGGTA −1114541 17 5 .3315 CACCACCGTTG −1131967 18 5 .3315 CAGAACCCACG −1180999 19 5 .3315 CCCTGAGTCCA −1434325 20 5 .3315 CCCTGGGTTCT −1436408 21 5 .3315 GCCCGGGAATA −2451981 22 5 .3315 GCGGCGGATGG −2529851 23 5 .3315 GGAAGCCACTT −2630944 24 5 .3315 TAAAGAGGCCG −3154583 25 5 .3315 TATGTCAAGCT −3388456 26 5 .3315 TGGGTTGTCTA −3849949 27 5 .3315 TGTAGTGTAAT −3878596 28 5 .3315 TTCAGTGGACC −4009606 29 5 .3315 TTGGTGAAGGA −4110377 30 4 .2652 AGAAACCAATA −525581 31 4 .2652 AGTGAGGAAGA −756233 32 4 .2652 ATAATACATAA −799025 33 4 .2652 ATACTGAAGCC −817190 34 4 .2652 ATTCTCCAGTG −1013039 35 4 .2652 CAAACTCTCAC −1056210 36 4 .2652 CAGTCACCAAC −1233218 37 4 .2652 CCCACAAGGTA −1380525 38 4 .2652 CCTTGCTCAAT −1566532 39 4 .2652 CCTTTGAGATC −1570958 40 4 .2652 CTAGTCTTTGT −1882108 41 4 .2652 CTGAACATCTC −1967326 42 4 .2652 GGCAAGCCCCA −2689365 43 4 .2652 GGCCTGGCTTA −2718333 44 4 .2652 TCCCCGTACAT −3496724 45 4 .2652 TCCCTATTAAG −3503043 46 4 .2652 TCTTCTCACAA −3661073 47 4 .2652 TGGCCCAAATT −3822608 48 3 .1989 AAGGTGGAAGA −178697 49 3 .1989 ACATCATAGAT −316196 50 3 .1989 AGGAAGGCGGC −658026 51 3 .1989 CAAGTGGAAAA −1096193 52 3 .1989 CACGCTCCCGG −1154395 53 3 .1989 CAGGCCACACA −1217605 54 3 .1989 CCAGAACAGAC −1343778 55 3 .1989 CCCAGAGCACT −1385032 56 3 .1989 CCCTAAACTGA −1425529 57 3 .1989 CCTGATCTTTA −1543677 58 3 .1989 CCTTTAATCCC −1568982 59 3 .1989 CTCAACAGCAA −1901713 60 3 .1989 CTCCTGGACAC −1931794 61 3 .1989 CTGGCTTTCAG −2006995 62 3 .1989 GACTTTGGAAA −2227841 63 3 .1989 GAGCGTTTTGG −2256891 64 3 .1989 GATGACACCAG −2327635 65 3 .1989 GCTGCAGTTGA −2593529 66 3 .1989 GCTGCCCTCCA −2594261 67 3 .1989 GTCTGCTGATG −3008399 68 3 .1989 GTGGAGGCGCC −3050086 69 3 .1989 GTGGGCGTGTA −3057389 70 3 .1989 TAAACCTGCTA −3151773 71 3 .1989 TATCCCACGCC −3363942 72 3 .1989 TCGGTTTCTGC −3587962 73 3 .1989 TCTCACCACCC −3622166 74 3 .1989 TGACCCCGGGA −3691945 75 3 .1989 TGCACAGTGCT −3740392 76 3 .1989 TGGTCTGGTCC −3858102 77 3 .1989 TGTAACAGGAC −3867810 78 3 .1989 TTCAGGTGGTT −4008880 79 3 .1989 TTGGCTGCCCA −4103765 80 2 .1326 AAAACAGTGGC −4842 81 2 .1326 AAAGCAGTGCT −37608 82 2 .1326 AAGAGGCAAGA −141577 83 2 .1223 AAGCAACAGGT −147756 84 2 .1326 AAGGTCGAGCT −177704 85 2 .1326 ACAGAACTCTT −295392 86 2 .1326 ACCTTGGAAGG −391691 87 2 .1326 ACTCTTTGTTT −491456 88 2 .1326 ACTGGCTGGGC −501674 89 2 .1326 ACTTATTATGC −511802 90 2 .1326 AGAACCATTAA −529649 91 2 .1326 AGACCCTCTCA −546677 92 2 .1326 AGCAATTCAAA −593729 93 2 .1326 ATCAACACCGC −853082 94 2 .1326 ATCCGAAAGAT −876580 95 2 .1326 CACCACCACAG −1131795 96 2 .1326 CACCTTGGTGC −1146554 97 2 .1326 CACGGGACCAC −1157202 98 2 .1326 CACTGACCTCC −1171830 99 2 .1326 CATTATGGGTG −1298095 100 2 .1326 CCCAATGGCCC −1379990 101 2 .1326 CCCGGACTTAC −1417714 102 2 .1326 CCCGTAGCCCC −1421910 103 2 .1326 CCTACAGTTGA −1512185 104 2 .1326 CCTCGGAAAAT −1533956 105 2 .1326 CCTGTGTGAAA −1555329 106 2 .1326 CGCCTGCTAGC −1669578 107 2 .1326 CGCTGGTTCCA −1698773 108 2 .1326 CGGTTCCACCC −1766678 109 2 .1326 CTAATAAAGCC −1847334 110 2 .1326 CTACCAGGATA −1856141 111 2 .1326 CTGCTATCCGA −1995609 112 2 .1326 CTGCTTTGTGC −1998778 113 2 .1326 CTGGGCGTGTC −2008814 114 2 .1326 CTGTAGGTGAT −2018020 115 2 .1326 CTGTGCCCTCC −2024822 116 2 .1326 CTTAAGGATCC −2034230 117 2 .1326 GAATCTGAAGT −2153996 118 2 .1326 GAATGCAGGGA −2155689 119 2 .1326 GAGGAGAAGAA −2263073 120 2 .1326 GATGCATAGTG −2331439 121 2 .1326 GATGTGACCAC −2340946 122 2 .1326 GATTTCTGTCT −2357176 123 2 .1326 GCACCGAACAC −2381842 124 2 .1326 GCATACGGCGC −2410138 125 2 .1326 GCATTGCATCT −2423096 126 2 .1326 GCCAAGGGTCA −2427573 127 2 .1326 GCCAAGTGGAG −2427811 128 2 .1326 GCCTAATGTAC −2474930 129 2 .1326 GCCTCGGGGGA −2480809 130 2 .1326 GCGACGCGGGC −2496938 131 2 .1326 GCGGCGGGATG −2529935 132 2 .1326 GCTCAGGATTC −2574910 133 2 .1326 GCTGGCAGACG −2598023 134 2 .1326 GCTTGCTTCCT −2615256 135 2 .1326 GGAAGGTGTCT −2632632 136 2 .1326 GGATTTGGCTT −2686624 137 2 .1326 GGGAGCTGTGC −2762682 138 2 .1326 GGGGAAATCGC −2785498 139 2 .1326 GGGGCTCAGCC −2792742 140 2 .1326 GGTGAGCCTGA −2853241 141 2 .1326 GGTGGGACACA −2861125 142 2 .1326 GTAAGCATAAA −2892993 143 2 .1326 GTCTGGGGGGA −3009193 144 2 .1326 GTGGTAGGCTA −3060381 145 2 .1326 GTTGCTGAGAA −3120673 146 2 .1326 GTTGGGGGGGG −3123883 147 2 .1326 TAACTGACAAT −3176516 148 2 .1326 TATACAATACA −3346629 149 2 .1326 TCCACTGTGCA −3481317 150 2 .1326 TCTGGACGCGG −3645851 151 2 .1326 TGAAACACTGT −3671164 152 2 .1326 TGACCCCGGGT −3691948 153 2 .1326 TGCCTGTGATA −3767181 154 2 .1326 TGGATCCTGAG −3814883 155 2 .1326 TGGGCAAAGCC −3837990 156 2 .1326 TGGTGACAAAA −3858689 157 2 .1326 TGTGCCAAGTG −3904559 158 2 .1326 TGTTCATCTTG −3920767 159 2 .1326 TTCAGCTCGAG −4007779 160 2 .1326 TTGCTGCAGTG −4094255 161 Tags included in this report = 161 DataBase Link Database c: 16 1.061% GGCTTCGGTCT (SEQ ID NO:1) −2750136 Noted Tags = 0 Collected Tags = 0 13 .862% AGAGCGAAGTG (SEQ ID NO:2) −563247 Noted Tags = 1 Collected Tags = 1 GCGGAA, Class A, U93862, Mus musculus ribosomal protein L41 mRNA, complete 12 .7957% AGCAGTCCCCT (SEQ ID NO:3) −601432 Noted Tags = 4 Collected Tags = 0 12 .7957% GTGGCTCACAA (SEQ ID NO:4) −3054865 Noted Tags = 207 Collected Tags = 56 CCATCC, Class A, AB0046, Mus musculus mRNA for Rab33B, complete cds. CCATCT, Class C, L29190, Mouse MHC class I H2-D transplantation antigen gen CCATCT, Class A, U20225, Mus musculus adenylosuccinate lyase (adl) mRNA, co CCATCT, Class A, X93168, M. musculus mRNA for cannabinoid receptor 2. CCATCT, Class A, X56974, M. musculus mRNA for external transcribed spacer (p CCATCT, Class C, X52915, M. musculus gene for H-2D(q) antigen, partial 3′c CCATCT, Class C, V00751, Mouse gene H-2Ld coding for a transplantation anti CCATCT, Class C, X52916, M. musculus H-2L(q) gene for H-2L(q) antigen. 3′p CCATCT, Class C, U06244, Mus musculus interferon alpha/beta receptor (IFNAR CCATCC, Class C, X64716, M. musculus NKR-P1 2 gene for natural killer cell r 10 .6631% AGGCAGACAGT (SEQ ID NO:5) −673868 Noted Tags = 2 Collected Tags = 2 TGCTGT, Class A, X13661, Mouse mRNA for elongation factor 1-alpha (EF 1-alp TGCTGT, Class A, M22432, Mus musculus protein synthesis elongation factor T 9 .5968% GCTGGCCCTTC (SEQ ID NO:6) −2598270 Noted Tags = 0 Collected Tags = 0 8 .5305% AGGTCGGGTGG (SEQ ID NO:7) −711355 Noted Tags = 1 Collected Tags = 0 8 .5305% CGCCGCCGGCT (SEQ ID NO:8) −1664424 Noted Tags = 1 Collected Tags = 0 7 .4641% CTGCTCAGGCT (SEQ ID NO:9) −1995944 Noted Tags = 1 Collected Tags = 1 TAGGAG, Class A, M13806, Mouse keratin (epidermal) type I mRNA, clone pkScc 7 .4641% TGGGCATCCAC (SEQ ID NO:10) −3838802 Noted Tags = 0 Collected Tags = 0 6 .3978% AAGGAAATGGG (SEQ ID NO:11) −164075 Noted Tags = 0 Collected Tags = 0 6 .3978% ATACTGACATT (SEQ ID NO:12) −817232 Noted Tags = 4 Collected Tags = 0 6 .3978% CCTTTGTGACT (SEQ ID NO:13) −1571720 Noted Tags = 0 Collected Tags 0 6 .3978% GAACATTGCAC (SEQ ID NO:14) −2117522 Noted Tags = 3 Collected = Tags 2 CACACG, Class A, X12697, Mouse p2-4 mRNA for SPARC/osteonectin (SPARC = sec CACACG, Class C, M20691, Mouse osteonectin (Sparc) gene, exon 9. 6 .3978% GGGAAGGCGGC (SEQ ID NO:15) −2755178 Noted Tags = 2 Collected Tags = 1 ACGTCT, Class A, M88335, M. musculus mRNA sequence. 5 .3315% ATGACTGATAG (SEQ ID NO:16) −925235 Noted Tags = 4 Collected Tags = 0 5 .3315% CACAAACGGTA (SEQ ID NO:17) −1114541 Noted Tags = 0 Collected Tags = 0 5 .3315% CACCACCGTTG (SEQ ID NO:18) −1131967 Noted Tags = 1 Collected Tags = 1 CCTTCA, Class C, M21460, Mouse surfeit locus surfeit 3 protein gene, exon 6 5 .3315% CAGAACCCACG (SEQ ID NO:19) −1180999 Noted Tags = 2 Collected Tags = 2 ACAGTA, Class C, M76762, Mus musculus ribosomal protein (Ke-3) gene, exons ACAGTA, Class A, M76763, Mus musculus ribosomal protein (Ke-3) mRNA, complete 5 .3315% CCCTGAGTCCA (SEQ ID NO:20) −1434325 Noted Tags = 2 Collected Tags = 2 CCCCGG, Class A, X03672, Mouse cytoskeletal mRNA for beta-actin. CCCCGG, Class A, J04181, Mouse A-X actin mRNA, complete cds. 5 .3315% CCCTGGGTTCT (SEQ ID NO:21) −1436408 Noted Tags = 2 Collected Tags = 1 GCCCGC, Class A, J04716, Mouse ferritin light chain, complete cds. 5 .3315% GCCCGGGAATA (SEQ ID NO:22) −2451981 Noted Tags = 1 Collected Tags = 1 AATTCA, Class A, J05277, Mouse hexokinase mRNA, complete cds. 5 .3315% GCGGCGGATGG (SEQ ID NO:23) −2529851 Noted Tags = 6 Collected Tags = 5 AGACTT, Class C, A27894, Coding sequence for GBP. AGACTT, Class A, X53067, Mouse mRNA for 14KDa lectin. AGACTT, Class A, X15986, Mouse 3′mRNA for beta-galactoside specific lectin AGACTT, Class A, X66532, M. musculus mRNA for L14 lectin. AGACTT, Class A, M57470, Murine beta-galactoside binding protein mRNA, comp 5 .3315% GGAAGCCACTT (SEQ ID NO:24) −2630944 Noted Tags = 0 Collected Tags = 0 5 .3315% TAAAGAGGCCG (SEQ ID NO:25) −3154583 Noted Tags = 1 Collected Tags = 1 TTTTGT, Class A, U67770, Mus musculus ribosomal protein S26 (RPS26) mRNA, c 5 .3315% TATGTCAAGCT (SEQ ID NO:26) −3388456 Noted Tags = 1 Collected Tags = 1 GGTGGA, Class A, X15962, Mouse mRNA for ribosomal protein S12. 5 .3315% TGGGTTGTCTA (SEQ ID NO:27) −3849949 Noted Tags = 1 Collected Tags = 1 AAAATA, Class A, X06407, Mouse mRNA for 21 kd polypeptide under translation 5 .3315% TGTAGTGTAAT (SEQ ID NO:28) −3878596 Noted Tags = 1 Collected Tags = 1 AAAGGT, Class A, X73829, M. musculus mRNA for ribosomal protein S8. 5 .3315% TTCAGTGGACC (SEQ ID NO:29) −4009606 Noted Tags = 0 Collected Tags = 0 5 .3315% TTGGTGAAGGA (SEQ ID NO:30) −4110377 Noted Tags = 2 Collected Tags = 0 4 .2652% AGAAACCAATA (SEQ ID NO:31) −525581 Noted Tags = 2 Collected Tags = 1 CGAACA, Class A, V00830, Mouse mRNA encoding epidermal keratin subunit. 4 .2652% AGTGAGGAAGA (SEQ ID NO:32) −756233 Noted Tags = 1 Collected Tags = 0 4 .2652% ATAATACATAA (SEQ ID NO:33) −799025 Noted Tags = 4 Collected Tags = 0 4 .2652% ATACTGAAGCC (SEQ ID NO:34) −817190 Noted Tags = 1 Collected Tags = 1 CCACTT, Class A, U28917, Mus musculus 60S ribosomal protein (A52) mRNA, com 4 .2652% ATTCTCCAGTG (SEQ ID NO:35) −1013039 Noted Tags = 1 Collected Tags = 0 4 .2652% CAAACTCTCAC (SEQ ID NO:36) −1056210 Noted Tags = 2 Collected Tags = 2 AGCGAT, Class A, X04017, Mouse mRNA for cysteine-rich glycoprotein SPARC. AGCGAT, Class C, M20692, Mouse osteonectin (Sparc) gene, exon 10. 4 .2652% CAGTCACCAAC (SEQ ID NO:37) −1233218 Noted Tags = 0 Collected Tags = 0 4 .2652% CCCACAAGGTA (SEQ ID NO:38) −1380525 Noted Tags = 0 Collected Tags = 0 4 .2652% CCTTGCTCAAT (SEQ ID NO:39) −1566532 Noted Tags = 2 Collected Tags = 1 TAAAA, Class A, M59470, Mouse cystatin C mRNA, complete cds. 4 .2652% CCTTTGAGATC (SEQ ID NO:40) −1570958 Noted Tags = 1 Collected Tags = 1 ATCCAC, Class A, U78085, Mus musculus ribosomal protein S5 mRNA, complete c 4 .2652% CTAGTCTTTGT (SEQ ID NO:41) −1882108 Noted Tags = 1 Collected = Tags ACACAA, Class A, L31609, Mus musculus (clone mcori-1ck9)S29 ribosomal prot 4 .2652% CTGAACATCTC (SEQ ID NO:42) −1967326 Noted Tags = 1 Collected Tags = 1 CCCCTT, Class A, X15267, Mouse mRNA for acidic ribosomal phosophoprotein PO 4 .2652% GGCAAGCCCCA (SEQ ID NO:43) −2689365 Noted Tags = 2 Collected Tags = 1 GCGTCT, Class A, U 12403, Mus musculus Csa-19 mRNA, complete cds. 4 .2652% GGCCTGGCTTA (SEQ ID NO:44) −2718333 Noted Tags = 0 Collected Tags = 0 4 .2652% TCCCCGTACAT (SEQ ID NO:45) −3496724 Noted Tags = 0 Collected Tags = 0 4 .2652% TCCCTATTAAG (SEQ ID NO:46) −3503043 Noted Tags = 0 Collected Tags = 0 4 .2652% TCTTCTCACAA (SEQ ID NO:47) −3661073 Noted Tags = 0 Collected Tags = 0 4 .2652% TGGCCCAAATT (SEQ ID NO:48) −3822608 Noted Tags = 2 Collected Tags = 1 TATGCC, Class C, M11409, Mouse S16 ribosomal protein processed pseudogene. 3 .1989% AAGGTGGAAGA (SEQ ID NO:49) −178697 Noted Tags = 0 Collected Tags = 0 3 .1989% ACATCATAGAT (SEQ ID NO:50) −316196 Noted Tags = 1 Collected Tags = 1 GACATC, Class A, L04280, Mus musculus ribosomal protein (Rpl12) mRNA, compl 3 .1989% AGGAAGGCGGC (SEQ ID NO:51) −658026 Noted tags = 0 Collected Tags = 0 3 .1989% CAAGTGGAAAA (SEQ ID NO:52) −1096193 Noted Tags = 0 Collected Tags = 0 3 .1989% CACGCTCCCGG (SEQ ID NO:53) −1154395 Noted Tags = 0 Collected Tags = 0 3 .1989% CAGGCCACACA (SEQ ID NO:54) −1217605 Noted Tags = 1 Collected Tags = 1 AGAGCC, Class A, AF0305, Mus musculus ATP synthase beta-subunit (beta-F1 AT 3 .1989% CCAGAACAGAC (SEQ ID NO:55) −1343778 Noted Tags = 3 Collected Tags = 0 4 .2447% CCCAGAGCACT (SEQ ID NO:56) −1385032 Noted Tags = 1 Collected Tags = 1 GGGTTG, Class A, M10937, Mouse epidermal 67-kDa type II keratin mRNA. 3 .1989% CCCTAAACTGA (SEQ ID NO:57) −1425529 Noted Tags = 0 Collected Tags = 0 3 .1989% CCTGATCTTTA (SEQ ID NO:58) −1543677 Noted Tags = 2 Collected Tags = 2 CTTCTA, Class A, X06406, Mouse mRNA for translational controlled 40 kDa pol CTTCTA, Class A, J02870, Mouse laminin receptor mRNA, complete cds. 3 .1989% CCTTTAATCCC (SEQ ID NO:59) −1568982 Noted Tags = 58 Collected Tags = 9 AGCACT, Class A, D42051, Mus musculus mRNA for Glutamate Decarboxylase, com AGAGGC, Class A, X79508, M. musculus (C57BL/10) CW37 mRNA, B1 repeat. AGCACT, Class A, U03421, Mus musculus interleukin-11 mRNA, complete cds. AGTTAC, Class C, U59807, Mus musculus cystatin B (Stfb) gene, complete cds. AGCACT, Class A, M58288, Mus musculus granulocyte colony-stimulating factor AGCACC, Class C, M14361, Mouse Ig germline kappa-chain V-region gene V-Ser, AGCAAT, Class C, M93320, Mus musculus DNA fragment, L1 repeat family region AGCCCT, Class C, J00632, mouse b1 ubiquitous repeat (copy c) mrna and flank AGGACT, Class A, L08394, Mus musculus betacellulin (bcn) mRNA, complete cds 3 .1989% CTCAACAGCAA (SEQ ID NO:60) −1901713 Noted Tags = 0 Collected Tags = 0 3 .1989% CTCCTGGACAC (SEQ ID NO:61) −1931794 Noted Tags = 1 Collected Tags = 1 CTGGGA, Class A, J04953, Mouse gelsolin gene, complete cds. 3 .1989% CTGGCTTTCAG (SEQ ID NO:62) −2006995 Noted Tags = 0 Collected Tags = 0 3 .1989% GACTTTGGAAA (SEQ ID NO:63) −2227841 Noted Tags = 6 Collected Tags = 3 ACATTT, Class A, U03419, Mus musculus alpha-1 type I procollagen mRNA, part ACATTT, Class C, X57981, Mouse gene for pro alpha1 (I) collagen chain (COL1 ACATTT, Class A, U08020, Mus musculus FVB/N collagen pro-alpha-1 type I cha 3 .1989% GAGCGTTTTGG (SEQ ID NO:64) −2256891 Noted Tags = 2 Collected Tags = 1 GTCCAG, Class A, X52803, Mouse mRNA for cyclophilin (EC 5.2.1.8). 3 .1989% GATGACACCAG (SEQ ID NO:65) −2327635 Noted Tags = 1 Collected Tags = 1 CCGCTC, Class A, U11248, Mus musculus C57BL/6J ribosomal protein S28 mRNA, 3 .1989% GCTGCAGTTGA (SEQ ID NO:66) −2593529 Noted Tags = 0 Collected Tags = 0 3 .1989% GCTGCCCTCCA (SEQ ID NO:67) −2594261 Noted Tags = 5 Collected Tags = 0 3 0.1989% GTCTGCTGATG (SEQ ID NO:68) −3008399 Noted Tags = 2 Collected Tags = 2 GCCAGA, Class A, X75313, M. musculus (C57BL/6) GB-like mRNA. GCCAGA, Class A, D29802, Mouse mRNA for G protein beta subunit homologue, c 3 .1989% GTGGAGGCGCC (SEQ ID NO:69) −3050086 Noted Tags = 0 Collected Tags = 0 3 .1989% GTGGGCGTGTA (SEQ ID NO:70) −3057389 Noted Tags = 1 Collected Tags = 1 CAACGG, Class A, M33330, Mouse insulinoma (rig) mRNA, complete cds. 3 .1989% TAAACCTGCTA (SEQ ID NO:71) −3151773 Noted Tags = 0 Collected Tags = 0 3 .1989% TATCCCACGCC (SEQ ID NO:72) −3363942 Noted Tags = 0 Collected Tags = 0 3 .1989% TCGGTTTCTGC (SEQ ID NO:73) −3587962 Noted Tags = 0 Collected Tags = 0 3 .1989% TCTCACCACCC (SEQ ID NO:74) −3622166 Noted Tags = 0 Collected Tags = 0 3 .1989% TGACCCCGGGA (SEQ ID NO:75) −3691945 Noted Tags = 0 Collected Tags = 0 3 .1989% TGCACAGTGCT (SEQ ID NO:76) −3740392 Noted Tags = 5 Collected Tags = 4 GAGCAA, Class A, X05835, Mouse mRNA for placental calcium-binding protein. GAGCAA, Class A, X16190, Mouse mts1 gene. GAGCAA, Class A, Z36947, Murine retrovirus RNA containing parts of mts1 of GAGCAA, Class A, D00208, Mus musculus mRNA for pEL98 protein, complete cds. 3 .1989% TGGTCTGGTCC (SEQ ID NO:77) −3858102 Noted Tags = 0 Collected Tags = 0 3 .1989% TGTAACAGGAC (SEQ ID NO:78) −3867810 Noted Tags = 1 Collected Tags = 1 TGCTAT, Class A, X04648, Mouse mRNA for IgG1/IgG2b Fc receptor (FcR). 3 .1989% TTCAGGTGGTT (SEQ ID NO:79) −4008880 Noted Tags = 1 Collected Tags = 1 TCTTCT, Class A, X73607, M. musculus mRNA for tropomyosin 5 (3′UTR). 3 .1989% TTGGCTGCCCA (SEQ ID NO:80) −4103765 Noted Tags = 1 Collected Tags = 1 GGATGT, Class C, Y08307, M. musculus mitochondrial mRNA for ribosomal protei 2 .1326% AAAACAGTGGC (SEQ ID NO:81) −4842 Noted Tags = 1 Collected Tags = 1 CGGTGG, Class A, X73331, M. musculus mRNA for ribosomal protein L37a. 2 .1326% AAAGCAGTGCT (SEQ ID NO:82) −37608 Noted Tags = 0 Collected Tags = 0 2 .1326% AAGAGGCAAGA (SEQ ID NO:83) −141577 Noted Tags = 0 Collected Tags = 0 2 .1326% AAGCAACAGGT (SEQ ID NO:84) −147756 Noted Tags = 0 Collected Tags = 0 2 .1326% AAGGTCGAGCT (SEQ ID NO:85) −177704 Noted Tags = 0 Collected Tags = 0 2 .1326% ACAGAACTCTT (SEQ ID NO:86) −295392 Noted Tags = 1 Collected Tags = 1 CTCAAT, Class A, AJ0023, Mus musculus mRNA for annexin VIII. 2 .1326% ACCTTGGAAGG (SEQ ID NO:87) −391691 Noted Tags = 1 Collected Tags = 0 2 .1326% ACTCTTTGTTT (SEQ ID NO:88) −491456 Noted Tags = 0 Collected Tags = 0 2 .1326% ACTGGCTGGGC (SEQ ID NO:89) −501674 Noted Tags = 0 Collected Tags = 0 2 .1326% ACTTATTATGC (SEQ ID NO:90) −511802 Noted Tags = 0 Collected Tags = 0 2 .1326% AGAACCATTAA (SEQ ID NO:91) −529649 Noted Tags = 0 Collected Tags = 0 2 .1326% AGACCCTCTCA (SEQ ID NO:92) −546677 Noted Tags = 0 Collected Tags = 0 2 .1326% AGCAATTCAAA (SEQ ID NO:93) −593729 Noted Tags = 6 Collected Tags = 2 CAATTA, Class C, U09637, Mus musculus domesticus mitochondrion NADH dehydro CAATTA, Class C, U09638, Mus musculus musculus mitochondrion NADH dehydroge 2 .1326% ATCAACACCGC (SEQ ID NO:94) −853082 Noted Tags = 2 Collected Tags = 2 AACCTT, Class A, Y00703, Mouse uncoupled 549 cells mRNA for stimulatory GTP AACCTT, Class A, M13964, Mouse stimulatory G protein of adenylate cyclase, 2 .1326% ATCCGAAAGAT (SEQ ID NO:95) −876580 Noted Tags = 2 Collected Tags = 1 GAAGCT, Class A, L04128, Mus musculus ribosomal protein L18 (rpL18) mRNA, c 2 .1326% CACCACCACAG (SEQ ID NO:96) −1131795 Noted Tags = 1 Collected Tags = 1 GATCAA, Class A, X05021, Murine mRNA with homology to yeast L29 ribosomal p 2 .1326% CACCTTGGTGC (SEQ ID NO:97) −1146554 Noted Tags = 0 Collected Tags = 0 2 .1326% CACGGGACCAC (SEQ ID NO:98) −1157202 Noted Tags = 0 Collected Tags = 0 2 .1326% CACTGACCTCC (SEQ ID NO:99) −1171830 Noted Tags = 1 Collected Tags = 0 2 .1326% CATTATGGGTG (SEQ ID NO:100) −1298095 Noted Tags = 3 Collected Tags = 3 GCAAGA, Class A, U16818, Mus musculus UDP glucuronosyltransferase (UGT1-06) GCAAGA, Class A, L02333, Murine bilirubin/phenol family UDP glucuronosyltra GCAAGA, Class A, L27122, Mus musculus (A-1) bilirubin/phenol UDP-glucuronos 2 .1326% CCCAATGGCCC (SEQ ID NO:101) −1379990 Noted Tags = 3 Collected Tags = 3 AATAAA, Class A, X65582, M. musculus mRNA for alpha-2 collagen VI. AATAAA, Class A, X62332, M. musculus mRNA for alpha-2 collagen type VI, subu AATAAA, Class A, Z18272, Mus musculus collagen alpha 2 chain type VI. 2 .1326% CCCGGACTTAC (SEQ ID NO:102) −1417714 Noted Tags = 2 Collected Tags = 0 2 .1326% CCCGTAGCCCC (SEQ ID NO:103) −1421910 Noted Tags = 1 Collected Tags = 1 TTCCGA, Class A, M22479, Mouse tropomyosin isoform 2 mRNA, complete cds. 2 .1326% CCTACAGTTGA (SEQ ID NO:104) −1512185 Noted Tags = 3 Collected Tags = 3 TAATCT, Class C, K02241, Mouse alkali myosin light chains, exons 6 and 7 co TAATCT, Class A, K02242, Mouse alkali myosin light chains MLC1f and MLC3f 3 TAATCT, Class C, K02243, Mouse alkali myosin light chains MLC1f/MLC3f pseud 2 .1326% CCTCGGAAAAT (SEQ ID NO:105) −1533956 Noted Tags = 0 Collected Tags = 0 2 .1326% CCTGTGTGAAA (SEQ ID NO:106) −1555329 Noted Tags = 0 Collected Tags = 0 2 .1326% CGCCTGCTAGC (SEQ ID NO:107) −1669578 Noted Tags = 2 Collected Tags = 1 CAACCG, Class A, X58251, Mouse COL1A2 mRNA for pro-alpha-2(1) collagen. 2 .1326% CGCTGGTTCCA (SEQ ID NO:108) −1698773 Noted Tags = 2 Collected Tags = 0 2 .1326% CGGTTCCACCC (SEQ ID NO:109) −1766678 Noted Tags = 0 Collected Tags = 0 2 .1326% CTAATAAAGCC (SEQ ID NO:110) −1847334 Noted Tags = 3 Collected Tags = 2 ACTGTG, Class A, X65922, M. musculus fau mRNA. ACTGTG, Class A, D26610, Mouse mRNA for monoclonal nonspecific suppressor f 2 .1326% CTACCAGGATA (SEQ ID NO:111) −1856141 Noted Tags = 0 Collected Tags = 0 3 .1835% CTGCTATCCGA (SEQ ID NO:112) −1995609 Noted Tags = 1 Collected Tags = 1 GAGAAT, Class A, X83590, M. musculus mRNA for ribosomal protein L5, 3′end. 2 .1326% CTGCTTTGTGC (SEQ ID NO:113) −1998778 Noted Tags = 2 Collected Tags = 2 TGTACA, Class A, M13227, Mouse enkephalin mRNA. TGTACA, Class A, M55181, Mouse spermatogenic-specific proenkephalin mRNA, c 2 .1326% CTGGGCGTGTC (SEQ ID NO:114) −2008814 Noted Tags = 0 Collected Tags = 0 2 .1326% CTGTAGGTGAT (SEQ ID NO:115) −2018020 Noted Tags = 0 Collected Tags = 0 2 .1326% CTGTGCCCTCC (SEQ ID NO:116) −2024822 Noted Tags = 0 Collected Tags = 0 2 .1326% CTTAAGGATCC (SEQ ID NO:117) −2034230 Noted Tags = 0 Collected Tags = 0 2 .1326% GAATCTGAAGT (SEQ ID NO:118) −2153996 Noted Tags = 0 Collected Tags = 0 2 .1326% GAATGCAGGGA (SEQ ID NO:119) −2155689 Noted Tags = 0 Collected Tags = 0 2 .1326% GAGGAGAAGAA (SEQ ID NO:120) −2263073 Noted Tags = 2 Collected Tags = 1 AGCATT, Class A, Y00225, Murine mRNA for J1 protein, yeast ribosomal protein 2 .1326% GATGCATAGTG (SEQ ID NO:121) −2331439 Noted Tags = 1 Collected Tags = 0 2 .1326% GATGTGACCAC (SEQ ID NO:122) −2340946 Noted Tags = 0 Collected Tags = 0 2 .1326% GATTTCTGTCT (SEQ ID NO:123) −2357176 Noted Tags = 0 Collected Tags = 0 2 .1326% GCACCGAACAC (SEQ ID NO:124) −2381842 Noted Tags = 0 Collected Tags = 0 2 .1326% GCATACGGCGC (SEQ ID NO:125) −2410138 Noted Tags = 1 Collected Tags = 0 2 .1326% GCATTGCATCT (SEQ ID NO:126) −2423096 Noted Tags = 0 Collected Tags = 0 2 .1326% GCCAAGGGTCA (SEQ ID NO:127) −2427573 Noted Tags = 1 Collected Tags = 1 GAGGCT, Class A, L08651, Mus musculus large ribosomal subunit protein mRNA, 2 .1326% GCCAAGTGGAG (SEQ ID NO:128) −2427811 Noted Tags = 5 Collected Tags = 1 TTCCCA, Class A, M76131, Mouse elongation factor 2 (ef−2) mRNA, 3′end. 2 .1326% GCCTAATGTAC (SEQ ID NO:129) −2474930 Noted Tags = 1 Collected Tags = 1 ACAAAG, Class A, U93863, Mus musculus ribosomal protein L21 mRNA, complete 2 .1326% GCCTCGGGGGA (SEQ ID NO:130) −2480809 Noted Tags = 0 Collected Tags = 0 2 .1326% GCGACGCGGGC (SEQ ID NO:131) −2496938 Noted Tags = 0 Collected Tags 0 2 .1326% GCGGCGGGATG (SEQ ID NO:132) −2529935 Noted Tags = 0 Collected Tags = 0 2 .1326% GCTCAGGATTC (SEQ ID NO:133) −2574910 Noted Tags = 1 Collected Tags = 1 ATCTGA, Class A, X91824, M. musculus mRNA for SPRR1a protein. 2 .1326% GCTGGCAGACG (SEQ ID NO:134) −2598023 Noted Tags = 0 Collected Tags = 0 2 .1326% GCTTGCTTCCT (SEQ ID NO:135) −2615256 Noted Tags = 1 Collected Tags = 1 GGAGCA, Class A, X89650, M. musculus mRNA for Rab7 protein. 2 .1326% GGAAGGTGTCT (SEQ ID NO:136) −2632632 Noted Tags = 0 Collected Tags = 0 2 .1326% GGATTTGGCTT (SEQ ID NO:137) −2686624 Noted Tags = 0 Collected Tags = 0 2 .1326% GGGAGCTGTGC (SEQ ID NO:138) −2762682 Noted Tags = 0 Collected Tags = 0 2 .1326% GGGGAAATCGC (SEQ ID NO:139) −2785498 Noted Tags = 1 Collected Tags = 1 CAGCTT, Class A, Z48496, M. musculus mRNA for testis-specific thymosin beta- 2 .1326% GGGGCTCAGCC (SEQ ID NO:140) −2792742 Noted Tags = 2 Collected Tags = 0 2 .1326% GGTGAGCCTGA (SEQ ID NO:141) −2853241 Noted Tags = 1 Collected Tags = 1 AGCTTG, Class A, U20611, Mus musculus thioredoxin-dependent peroxide reduct 2 .1326% GGTGGGACACA (SEQ ID NO:142) −2861125 Noted Tags = 0 Collected Tags = 0 2 .1326% GTAAGCATAAA (SEQ ID NO:143) −2892993 Noted Tags = 0 Collected Tags = 0 2 .1326% GTCTGGGGGGA (SEQ ID NO:144) −3009193 Noted Tags = 0 Collected Tags = 0 2 .1326% GTGGTAGGCTA (SEQ ID NO:145) −3060381 Noted Tags = 0 Collected Tags = 0 2 .1326% GTTGCTGAGAA (SEQ ID NO:146) −3120673 Noted Tags = 2 Collected Tags = 2 GCGGCT, Class A, X75312, M. musculus (C57BL/6)QM mRNA. GCGGCT, Class A, M93980, Mouse 24.6 kda protein mRNA, complete cds. 2 .1326% GTTGGGGGGGG (SEQ ID NO:147) −3123883 Noted Tags = 0 Collected Tags = 0 2 .1326% TAACTGACAAT (SEQ ID NO:148) −3176516 Noted Tags = 1 Collected Tags = 0 2 .1326% TATACAATACA (SEQ ID NO:149) −3346629 Noted Tags = 0 Collected Tags = 0 2 .1326% TCCACTGTGCA (SEQ ID NO:150) −3481317 Noted Tags = 1 Collected Tags = 1 CGTGTG, Class C, L38580, Mus musculus galanin gene, exon 6 and complete cds 2 .1326% TCTGGACGCGG (SEQ ID NO:151) −3645851 Noted Tags = 2 Collected Tags = 2 AAAGCA, Class C, M33988, Mouse histone H2A.1 gene, complete cds. CAAGCA, Class C, M37736, Mouse replication-dependent histone H2A.1 gene. 2 .1326% TGAAACACTGT (SEQ ID NO:152) −3671164 Noted Tags = 2 Collected Tags = 2 , Class A, D86344, Mouse mRNA for Topoisomerase-inhibitor suppressed, T, Class A, D50465, Mouse MA-3 (apoptosis-related gene) mRNA, complete 2 .1326% TGACCCCGGGT (SEQ ID NO:153) −3691948 Noted Tags = 0 Collected Tags = 0 2 .1326% TGCCTGTGATA (SEQ ID NO:154) −3767181 Noted Tags = 0 Collected Tags = 0 2 .1326% TGGATCCTGAG (SEQ ID NO:155) −3814883 Noted Tags = 21 Collected Tags = 4 AACTTC, Class C, V00742, Fragment of the mouse gene for epsilon-globin Y3 ( AACTTC, Class A, M19236, Mouse beta-globin gene. AACTTC, Class C, J00414, Mouse beta-globin epsilon y3 gene, exon 2. AACTTC, Class C, M10688, Mouse beta-globin gene with intron boundary. 2 .1326% TGGGCAAAGCC (SEQ ID NO:156) −3837990 Noted Tags = 0 Collected Tags = 0 2 .1326% TGGTGACAAAA (SEQ ID NO:157) −3858689 Noted Tags = 0 Collected Tags = 0 2 .1326% TGTGCCAAGTG (SEQ ID NO:158) −3904559 Noted Tags = 0 Collected Tags = 0 2 .1326% TGTTCATCTTG (SEQ ID NO:159) −3920767 Noted Tags = 2 Collected Tags = 1 TTTTAA, Class C, X57983, Mouse gene for pro alpha1 (III) collagen chain (CO 2 .1326% TTCAGCTCGAG (SEQ ID NO:160) −4007779 Noted Tags = 0 Collected Tags = 0 2 .1326% TTGCTGCAGTG (SEQ ID NO:161) −4094255 Noted Tags = 0 Collected Tags = 0

TABLE 13 SAGE Analysis of MRL Mice Five Days After Wounding SearchName = C5BANK FileName = c:\sage\data\c5data.2\tagbank.bag Anchoring Enzyme = N1aIII-CATG Tag Length = 11 DiTag Length = 24 Total Files = 67 Total Tags = 1790 Total Duplicate Dimers = 0 Tag Abundance Report Total tags after excluding tags = 1790 Count Percent Tag Sequence −Tag BaseFour Number SEQ ID NO: 23 1.2849 CGGTCCAGGGA −1758377 162 18 1.0055 GTGGCTCACAA −3054865 163 16 .8938 GGCTTCGGTCT −2750136 164 14 .7821 AGGTCGGGTGG −711355 165 13 .7262 CACAAACGGTA −1114541 166 13 .7262 TGGGTTGTCTA −3849949 167 12 .6703 CGCCGCCGGCT −1664424 168 12 .6703 CTGCTCAGGCT −1995944 169 12 .6703 GCTGGCCCTTC −2598270 170 11 .6145 AGGCAGACAGT −673868 171 11 .6145 TTGGCTGCCCA −4103765 172 10 .5586 GCCCGGGAATA −2451981 173 9 .5027 AGAGCGAAGTG −563247 174 9 .5027 CAGAACCCACG −1180999 175 9 .5027 CATCGCCAGTG −1271087 176 9 .5027 CCCCAGCCAGT −1395020 177 8 .4469 AGCAGTCCCCT −601432 178 8 .4469 CAAACTCTCAC −1056210 179 8 .4469 CTAATAAAGCC −1847334 180 8 .4469 GGCAAGCCCCA −2689365 181 7 .391 CAAGGTGACAG −1093139 182 7 .391 CCAGAACAGAC −1343778 183 7 .391 CTGAACATCTC −1967326 184 7 .391 GCCTTTATGAG −2489571 185 7 .391 GGAAGCCACTT −2630944 186 7 .391 GTGAACGTGCC −3016422 187 7 .391 GTGGGCGTGTA −3057389 188 6 .3351 AACACCAAGCT −70696 189 6 .3351 CACCACCACAG −1131795 190 6 .3351 CACCACCGTTG −1131967 191 6 .3351 CACGCTCCCGG −1154395 192 6 .3351 CCCGTGTGCTC −1424286 193 6 .3351 CCTTGCTCAAT −1566532 194 6 .3351 CTGCTATCCGA −1995609 195 6 .3351 GAACATTGCAC −2117522 196 6 .3351 GGATTTGGCTT −2686624 197 6 .3351 GGGAAGGCGGC −2755178 198 6 .3351 GTCTGCTGATG −3008399 199 6 .3351 TCAGGCTGCCT −3450776 200 6 .3351 TGGATCCTGAG −3814883 201 5 .2793 CGCTGGTTCCA −1698773 202 5 .2793 CTCCTGGACAC −1931794 203 5 .2793 TATGTCAAGCT −3388456 204 5 .2793 TCGTGATTGTG −3597295 205 5 .2793 TTCAGTGGACC −4009606 206 4 .2234 AAGAGGCAAGA −141577 207 4 .2234 ATACTGACATT −817232 208 4 .2234 CAAGTGGAAAA −1096193 209 4 .2234 CCCAATGGCCC −1379990 210 4 .2234 CCTACCAAGAC −1512482 211 4 .2234 GATGACACCAG −2327635 212 4 .2234 GATTCCGTGAG −2348771 213 4 .2234 GCAGAGTGCGC −2395034 214 4 .2234 GCCAAGTGGAG −2427811 215 4 .2234 GCGGCGGATGG −2529851 216 4 .2234 GTGGAGGCGCC −3050086 217 4 .2234 TGCACAGTGCT −3740392 218 4 .2234 TGGATCAGTCT −3814584 219 4 .2234 TGGCTCGGTCA −3831477 220 4 .2234 TGTGCCAAGTG −3904559 221 4 .2234 TTCTTTGGTGA −4062905 222 3 .1675 AGTGAGGAAGA −756233 223 3 .1675 CACCTTGGTGC −1146554 224 3 .1675 CCCTGAGTCCA −1434325 225 3 .1675 CTACCACTCAA −1855953 226 3 .1675 CTGAGAGAGAA −1974817 227 3 .1675 CTGTAGACTGC −2017402 228 3 .1675 CTGTAGGTGAT −2018020 229 3 .1675 CTTGACACACA −2065477 230 3 .1675 GAGTCTCCCTG −2284895 231 3 .1675 GATGTGGCTGC −2341498 232 3 .1675 GCCGCTAGGCC −2464934 233 3 .1675 GCCTGTGGCCT −2485912 234 3 .1675 GCGCCCTCCCC −2512726 235 3 .1675 GGGGGCCCAGG −2794827 236 3 .1675 GTGTGGGCACT −3074632 237 3 .1675 GTGTTAACCAG −3076179 238 3 .1675 TAAAGAGGCCG −3154583 239 3 .1675 TGCTTATGATG −3797903 240 3 .1675 TGGTGACAAAA −3858689 241 3 .1675 TGTCAGTCTGT −3885948 242 3 .1675 TTCAGCTCGAG −4007779 243 2 .1117 AACAATTTGGG −69611 244 2 .1117 AACAGGTTCAA −76753 245 2 .1117 AAGCGCCTCAC −157138 246 2 .1117 AAGGAAATGGG −164075 247 2 .1117 AAGGTCTGCCT −178072 248 2 .1117 AAGGTGGAAGA −178697 249 2 .1117 ACAGTTCCAGA −310601 250 2 .1117 ACCCTCCTCCC −357846 251 2 .1117 AGAGGAAGCTG −565407 252 2 .1117 AGCAGGGATCC −600630 253 2 .1117 AGGAAGGCGGC −658026 254 Tags included in this report = 185 DataBase Link Database = c: 23 1.2849% CGGTCCAGGGA (SEQ ID NO:162) −1758377 Noted Tags = 0 Collected Tags = 0 18 1.0055% GTGGCTCACAA (SEQ ID NO:163) −3054865 Noted Tags = 207 Collected Tags = 56 CCATCC, Class A, AB0046, Mus musculus mRNA for Rab33B, complete cds. CCATCT, Class C, L29190, Mouse MHC class I H2-D transplantation antigen gen CCATCT, Class A, U20225, Mus musculus adenylosuccinate lyase (adl) mRNA, co CCATCT, Class A, X93168, M. musculus mRNA for cannabinoid receptor 2. CCATCT, Class A, X56974, M. musculus mRNA for external transcribed spacer (p CCATCT, Class C, X52915, M. musculus gene for H-2D(q) antigen, partial 3′ c CCATCT, Class C, V00751, Mouse gene H-2Ld coding for a transplantation anti CCATCT, Class C, X52916, M. musculus H-2L(q) gene for H-2L(q) antigen. 3′ p CCATCT, Class C, U06244, Mus musculus interferon alpha/beta receptor (IFNAR CCATCC, Class C, X64716, M. musculus NKR-P1 2 gene for natural killer cell r 16 .8938% GGCTTCGGTCT (SEQ ID NO:164) −2750136 Noted Tags = 0 Collected Tags = 0 14 .7821% AGGTCGGGTGG (SEQ ID NO:165) −711355 Noted Tags = 1 Collected Tags = 0 13 .7262% CACAAACGGTA (SEQ ID NO:166) −1114541 Noted Tags = 0 Collected Tags = 0 13 .7262% TGGGTTGTCTA (SEQ ID NO:167) −3849949 Noted Tags = 1 Collected Tags = 1 AAAATA, Class A, X06407, Mouse mRNA for 21 kd polypeptide under translation 12 .6703% CGCCGCCGGCT (SEQ ID NO:168) −1664424 Noted Tags = 1 Collected Tags = 0 12 .6703% CTGCTCAGGCT (SEQ ID NO:169) −1995944 Noted Tags = 1 Collected Tags = 1 TAGGAG, Class A, M13806, Mouse keratin (epidermal) type I mRNA, clone pkScc 12 .6703% GCTGGCCCTTC (SEQ ID NO:170) −2598270 Noted Tags = 0 Collected Tags = 0 11 .6145% AGGCAGACAGT (SEQ ID NO:171) −673868 Noted Tags = 2 Collected Tags = 2 TGCTGT, Class A, X13661, Mouse mRNA for elongation factor 1-alpha (EF 1-alp TGCTGT, Class A, M22432, Mus musculus protein synthesis elongation factor T 11 .6145% TTGGCTGCCCA (SEQ ID NO:172) −4103765 Noted Tags = 1 Collected Tags = 1 GGATGT, Class C, Y08307, M. musculus mitochondrial mRNA for ribosomal protei 10 .5586% GCCCGGGAATA (SEQ ID NO:173) −2451981 Noted Tags = 1 Collected Tags = 1 AATTCA, Class A, J05277, Mouse hexokinase mRNA, complete cds. 9 .5027% AGAGCGAAGTG (SEQ ID NO:174) −563247 Noted Tags = 1 Collected Tags = 1 GCGGAA, Class A, U93862, Mus musculus ribosomal protein L41 mRNA, complete 9 .5027% CAGAACCCACG (SEQ ID NO:175) −1180999 Noted Tags = 2 Collected Tags = 2 ACAGTA, Class C, M76762, Mus musculus ribosomal protein (Ke-3) gene, exons ACAGTA, Class A, M76763, Mus musculus ribosomal protein (Ke-3) mRNA, comple 9 .5027% CATCGCCAGTG (SEQ ID NO:176) −1271087 Noted Tags = 3 Collected Tags = 2 GGCAAA, Class A, M12414, Mouse apolipoprotein E mRNA, complete cds. GGCAAA, Class A, M73490, Mus musculus apolipoprotein E mRNA, 3′end. 9 .5027% CCCCAGCCAGT (SEQ ID NO:177) −1395020 Noted Tags = 2 Collected Tags = 1 GCCTAC, Class A, X76772, M. musculus mRNA for ribosomal protein S3. 8 .4469% AGCAGTCCCCT (SEQ ID NO:178) −601432 Noted Tags = 4 Collected Tags = 0 8 .4469% CAAACTCTCAC (SEQ ID NO:179) −1056210 Noted Tags = 2 Collected Tags = 2 AGCGAT, Class A, X04017, Mouse mRNA for cysteine-rich glycoprotein SPARC. AGCGAT, Class C, M20692, Mouse osteonectin (Sparc) gene, exon 10. 8 .4469% CTAATAAAGCC (SEQ ID NO:180) −1847334 Noted Tags = 3 Collected Tags = 2 ACTGTG, Class A, X65922, M. musculus fau mRNA. ACTGTG, Class A, D26610, Mouse mRNA for monoclonal nonspecific suppressor f 8 .4469% GGCAAGCCCCA (SEQ ID NO:181) −2689365 Noted Tags = 2 Collected Tags = 1 GCGTCT, Class A, U12403, Mus musculus Csa-19 mRNA, complete cds. 7 .391% CAAGGTGACAG (SEQ ID NO:182) −1093139 Noted Tags = 3 Collected Tags = 3 GCCGCT, Class A, M20632, Mouse LLRep3 protein mRNA from a repetitive elemen GCCGCT, Class C, M20633, Mouse LLRep3 protein pseudogene from a repetitive GCCGCT, Class C, M20634, Mouse LLRep3 protein pseudogene from a repetitive 7 .391% CCAGAACAGAC (SEQ ID NO:183) −1343778 Noted Tags = 3 Collected Tags = 0 7 .391% CTGAACATCTC (SEQ ID NO:184) −1967326 Noted Tags = 1 Collected Tags = 1 CCCCTT, Class A, X15267, Mouse mRNA for acidic ribosomal phosophoprotein PO 7 .391% GCCTTTATGAG (SEQ ID NO:185) −2489571 Noted Tags = 2 Collected Tags = 1 AAGAAA, Class A, X60289, M. musculus mRNA for ribosomal protein S24. 7 .391% GGAAGCCACTT (SEQ ID NO:186) −2630944 Noted Tags = Collected Tags = 0 7 .391% GTGAACGTGCC (SEQ ID NO:187) −3016422 Noted Tags = 0 Collected Tags = 0 7 .391% GTGGGCGTGTA (SEQ ID NO:188) −3057389 Noted Tags = 1 Collected Tags = 1 CAACGG, Class A, M33330, Mouse insulinoma (rig) mRNA, complete cds. 6 .3351% AACACCAAGCT (SEQ ID NO:189) −70696 Noted Tags = 2 Collected Tags = 2 GTCCCT, Class A, X74784, M. musculus mk2e mRNA. GTCCCT, Class A, M24151, Mouse keratin 70 kd type II mRNA, 3′end. 6 .3351% CACCACCACAG (SEQ ID NO:190) −1131795 Noted Tags = 1 Collected Tags = 1 GATCAA, Class A, X05021, Murine mRNA with homology to yeast L29 ribosomal p 6 .3351% CACCACCGTTG (SEQ ID NO:191) −1131967 Noted Tags = 1 Collected Tags = 1 CCTTCA, Class C, M21460, Mouse surfeit locus surfeit 3 protein gene, exon 6 6 .3351% CACGCTCCCGG (SEQ ID NO:192) −1154395 Noted Tags = 0 Collected Tags = 0 6 .3351% CCCGTGTGCTC (SEQ ID NO:193) −1424286 Noted Tags = 0 Collected Tags = 0 6 .3351% CCTTGCTCAAT (SEQ ID NO:194) −1566532 Noted Tags = 2 Collected Tags = 1 TAAAA, Class A, M59470, Mouse cystatin C mRNA, complete cds. 6 .3351% CTGCTATCCGA (SEQ ID NO:195) −1995609 Noted Tags = 1 Collected Tags = 1 GAGAAT, Class A, X83590, M. musculus mRNA for ribosomal protein L5, 3′end. 6 .3351% GAACATTGCAC (SEQ ID NO:196) −2117522 Noted Tags = 3 Collected Tags = 2 CACACG, Class A, X12697, Mouse p2-4 mRNA for SPARC/osteonectin (SPARC=sec CACACG, Class C, M20691, Mouse osteonectin (Sparc) gene, exon 9. 6 .3351% GGATTTGGCTT (SEQ ID NO:197) −2686624 Noted Tags = 0 Collected Tags = 0 6 .3351% GGGAAGGCGGC (SEQ ID NO:198) −2755178 Noted Tags = 2 Collected Tags = 1 ACGTCT, Class A, M88335, M. musculus mRNA sequence. 6 .3351% GTCTGCTGATG (SEQ ID NO:199) −3008399 Noted Tags = 2 Collected Tags = 2 GCCAGA, Class A, X75313, M. musculus (C57BL/6) GB-like mRNA. GCCAGA, Class A, D29802, Mouse mRNA for G protein beta subunit homologue, c 6 .3351% TCAGGCTGCCT (SEQ ID NO:200) −3450776 Noted Tags = 7 Collected Tags = 6 TCATCT, Class A, X12812, Murine mRNA for macrophage ferritin heavy subunit. TCATCT, Class C, X52561, Mouse gene for ferritin H subunit. TCATCT, Class A, J03941, Mouse ferritin heavy chain (MFH) mRNA, complete cd TTATCT, Class A, M24509, Mouse ferritin heavy chain, complete cds. TTATCC, Class C, M73678, Mus musculus (clone pMHFY9) ferritin H pseudogene. TTATCT, Class C, M73679, Mus musculus (clone pMHFY1) ferritin H pseudogene. 6 .3351% TGGATCCTGAG (SEQ ID NO:201) −3814883 Noted Tags = 21 Collected Tags = 4 AACTTC, Class C, V00742, Fragment of the mouse gene for epsilon-globin Y3 ( AACTTC, Class A, M19236, Mouse beta-globin gene. AACTTC, Class C, J00414, Mouse beta-globin epsilon y3 gene, exon 2. AACTTC, Class C, M10688, Mouse beta-globin gene with intron boundary. 5 .2793% CGCTGGTTCCA (SEQ ID NO:202) −1698773 Noted Tags = 2 Collected Tags = 0 5 .2793% CTCCTGGACAC (SEQ ID NO:203) −1931794 Noted Tags = 1 Collected Tags = 1 CTGGGA, Class A, J04953, Mouse gelsolin gene, complete cds. 5 .2793% TATGTCAAGCT (SEQ ID NO:204) −3388456 Noted Tags = 1 Collected Tags = 1 GGTGGA, Class A, X15962, Mouse mRNA for ribosomal protein S12. 5 .2793% TCGTGATTGTG (SEQ ID NO:205) −3597295 Noted Tags = 3 Collected Tags = 3 CAGAAT, Class A, U52822, Mus musculus ornithine decarboxylase antizyme mRNA CAGAAT, Class C, U52823, Mus musculus ornithine decarboxylase antizyme gene CAGAAT, Class C, U84291, Mus musculus ornithine decarboxylase antizyme gene 5 .2793% TTCAGTGGACC (SEQ ID NO:206) −4009606 Noted Tags = 0 Collected Tags = 0 4 .2234% AAGAGGCAAGA (SEQ ID NO:207) −141577 Noted Tags = 0 Collected Tags = 0 4 .2234% ATACTGACATT (SEQ ID NO:208) −817232 Noted Tags = 4 Collected Tags = 0 4 .2234% CAAGTGGAAAA (SEQ ID NO:209) −1096193 Noted Tags = 0 Collected Tags = 0 4 .2234% CCCAATGGCCC (SEQ ID NO:210) −1379990 Noted Tags = 3 Collected Tags = 3 AATAAA, Class A, X65582, M. musculus mRNA for alpha-2 collagen VI. AATAAA, Class A, X62332, M. musculus mRNA for alpha-2 collagen type VI, subu AATAAA, Class A, Z18272, Mus musculus collagen alpha 2 chain type VI. 4 .2234% CCTACCAAGAC (SEQ ID NO:211) −1512482 Noted Tags = 0 Collected Tags = 0 4 .2234% GATGACACCAG (SEQ ID NO:212) −2327635 Noted Tags = 1 Collected Tags = 1 CCGCTC, Class A, U11248, Mus musculus C57BL/6J ribosomal protein S28 mRNA, 4 .2234% GATTCCGTGAG (SEQ ID NO:213) −2348771 Noted Tags = 0 Collected Tags = 0 4 .2234% GCAGAGTGCGC (SEQ ID NO:214) −2395034 Noted Tags = 2 Collected Tags = 1 CTGCTG, Class A, Y00348, Mouse mRNA for ribosomal protein S6. 4 .2234% GCCAAGTGGAG (SEQ ID NO:215) −2427811 Noted Tags = 5 Collected Tags = 1 TTCCCA, Class A, M76131, Mouse elongation factor 2 (ef-2) mRNA, 3′end. 4 .2234% GCGGCGGATGG (SEQ ID NO:216) −2529851 Noted Tags = 6 Collected Tags = 5 AGACTT, Class C, A27894, Coding sequence for GBP. AGACTT, Class A, X53067, Mouse mRNA for 14KDa lectin. AGACTT, Class A, X15986, Mouse 3′mRNA for beta-galactoside specific lectin AGACTT, Class A, X66532, M. musculus mRNA for L14 lectin. AGACTT, Class A, M57470, Murine beta-galactoside binding protein mRNA, comp 4 .2234% GTGGAGGCGCC (SEQ ID NO:217) −3050086 Noted Tags = 0 Collected Tags = 0 4 .2234% TGCACAGTGCT (SEQ ID NO:218) −3740392 Noted Tags = 5 Collected Tags = 4 GAGCAA, Class A, X05835, Mouse mRNA for placental calcium-binding protein. GAGCAA, Class A, X16190, Mouse mts1 gene. GAGCAA, Class A, Z36947, Murine retrovirus RNA containing parts of mts1 of GAGCAA, Class A, D00208, Mus musculus mRNA for pEL98 protein, complete cds. 4 .2234% TGGATCAGTCT (SEQ ID NO:219) −3814584 Noted Tags = 1 Collected Tags = 1 TTAAAA, Class A, M62952, Mus musculus ribosomal protein L19, complete cds. 4 .2234% TGGCTCGGTCA (SEQ ID NO:220) −3831477 Noted Tags = 6 Collected Tags = 3 CTTGGG, Class A, X13055, Murine mRNA for cytoplasmic gamma-actin. CTTGGG, Class C, X13056, Murine gamma-118-actin pseudogene. CTTGGG, Class A, M21495, Mouse cytoskeletal gamma-actin mRNA, complete cds. 4 .2234% TGTGCCAAGTG (SEQ ID NO:221) −3904559 Noted Tags = 0 Collected Tags = 0 4 .2234% TTCTTTGGTGA (SEQ ID NO:222) −4062905 Noted Tags = 1 Collected Tags = 0 3 .1675% AGTGAGGAAGA (SEQ ID NO:223) −756233 Noted Tags = 1 Collected Tags = 0 3 .1675% CACCTTGGTGC (SEQ ID NO:224) −1146554 Noted Tags = 0 Collected Tags = 0 3 .1675% CCCTGAGTCCA (SEQ ID NO:225) −1434325 Noted Tags = 2 Collected Tags = 2 CCCCGG, Class A, X03672, Mouse cytoskeletal mRNA for beta-actin. CCCCGG, Class A, J04181, Mouse A-X actin mRNA, complete eds. 3 .1675% CTACCACTCAA (SEQ ID NO:226) −1855953 Noted Tags = 0 Collected Tags = 0 3 .1675% CTGAGAGAGAA (SEQ ID NO:227) −1974817 Noted Tags = 0 Collected Tags = 0 3 .1675% CTGTAGACTGC (SEQ ID NO:228) −2017402 Noted Tags = 0 Collected Tags = 0 3 .1675% CTGTAGGTGAT (SEQ ID NO:229) −2018020 Noted Tags = 0 Collected Tags = 0 3 .1675% CTTGACACACA (SEQ ID NO:230) −2065477 Noted Tags = 0 Collected Tags = 0 3 .1675% GAGTCTCCCTG (SEQ ID NO:231) −2284895 Noted Tags = 2 Collected Tags = 2 GATTGT, Class C, X59747, Mouse Sm B gene for Sm B protein of U snRNP's, exo GATTGT, Class A, M58761, Mouse Sm-B protein gene, complete cds. 3 .1675% GATGTGGCTGC (SEQ ID NO:232) −2341498 Noted Tags = 0 Collected Tags = 0 3 .1675% GCCGCTAGGCC (SEQ ID NO:233) −2464934 Noted Tags = 0 Collected Tags = 0 3 .1675% GCCTGTGGCCT (SEQ ID NO:234) −2485912 Noted Tags = 0 Collected Tags = 0 3 .1675% GCGCCCTCCCC (SEQ ID NO:235) −2512726 Noted Tags = 1 Collected Tags = 1 TTGTCC, Class A, U88322, Mus musculus beta chemokine Exodus-2 mRNA, complet 3 .1675% GGGGGCCCAGG (SEQ ID NO:236) −2794827 Noted Tags = 1 Collected Tags = 1 TGTAGA, Class C, X03059, Mouse germline gene for T-cell receptor J-alpha 65 3 .1675% GTGTGGGCACT (SEQ ID NO:237) −3074632 Noted Tags = 1 Collected Tags = 1 GGATTT, Class A, X93035, M. musculus mRNA for BRP39 protein. 3 .1675% GTGTTAACCAG (SEQ ID NO:238) −3076179 Noted Tags = 0 Collected Tags = 0 3 .1675% TAAAGAGGCCG (SEQ ID NO:239) −3154583 Noted Tags = 1 Collected Tags = 1 TTTTGT, Class A, U67770, Mus musculus ribosomal protein 526 (RPS26) mRNA, c 3 .1675% TGCTTATGATG (SEQ ID NO:240) −3797903 Noted Tags = 0 Collected Tags = 0 3 .1675% TGGTGACAAAA (SEQ ID NO:241) −3858689 Noted Tags = 0 Collected Tags = 0 3 .1675% TGTCAGTCTGT (SEQ ID NO:242) −3885948 Noted Tags = 1 Collected Tags = 1 TTAACC, Class C, M21050, Mouse lysozyme M gene, exon 4. 3 .1675% TTCAGCTCGAG (SEQ ID NO:243) −4007779 Noted Tags = 0 Collected Tags = 0 2 .1117% AACAATTTGGG (SEQ ID NO:244) −69611 Noted Tags = 0 Collected Tags = 0 2 .1117% AACAGGTTCAA (SEQ ID NO:245) −76753 Noted Tags = 0 Collected Tags = 0 2 .1117% AAGCGCCTCAC (SEQ ID NO:246) −157138 Noted Tags = 0 Collected Tags = 0 2 .1117% AAGGAAATGGG (SEQ ID NO:247) −164075 Noted Tags = 0 Collected Tags = 0 2 .1117% AAGGTCTGCCT (SEQ ID NO:248) −178072 Noted Tags = 1 Collected Tags = 1 GAAAAC, Class A, D87896, Mus musculus phgpx mRNA for phospholipid hydropero 2 .1117% AAGGTGGAAGA (SEQ ID NO:249) −178697 Noted Tags = 0 Collected Tags = 0 2 .1117% ACAGTTCCAGA (SEQ ID NO:250) −310601 Noted Tags = 3 Collected Tags = 3 AGTGAT, Class A, AF0234, Mus musculus HS1-associating protein (mHAX-1s)mRN AGTGAT, Class A, AF0234, Mus musculus HS-1-associating protein HAX-1L(mHAX AGTGAT, Class A, X81444, M. musculus SIG-111 mRNA. 2 .1117% ACCCTCCTCCC (SEQ ID NO:251) −357846 Noted Tags = 3 Collected Tags = 2 CCCGCT, Class C, K01365, Mouse 18S-5.8S-28S rRNA gene internal transcribed CCCGCT, Class C, J00623, Mouse 185, 5.8S, 28S rRNA gene cluster (clone pMEB 2 .1117% AGAGGAAGCTG (SEQ ID NO:252) −565407 Noted Tags = 0 Collected Tags = 0 2 .1117% AGCAGGGATCC (SEQ ID NO:253) −600630 Noted Tags = 1 Collected Tags = 1 CCGTGC, Class A, X97490, M. musculus mRNA for PNG protein. 2 .1117% AGGAAGGCGGC (SEQ ID NO:254) −658026 Noted Tags = 0 Collected Tags = 0

TABLE 14 Accession Numbers for Wound Healing Genes x60671 Ezrin Villin 2; NF-2 (merlin) related filament/plasma Alf membrane associated protein M26391 Rb; pp105; Retinoblastoma susceptibility-associated Alm protein (tumor suppressor gene; cell cycle regulator) U52945 TSG101 tumor susceptibility protein Aln U54705 Tumor suppressor maspin A2a D14340 ZO-1; Tight junction protein; discs-large family member, A2d partially homologous to a dig-A tumor suppressor in Drosophila X51983 o-ErbA oncogene; thyroid hormone receptor A2g J04115 o-Jun proto-oncogene (transcription factor AP-1 A2i component) X83974 RNA polymerase I termination factor TTF-1 A2j X01023 c-myc proto-oncogene protein A2l U51866 Casein kinase II (alpha subunit) A3n M13945 Pim-1 proto-oncogene A4a X68932 c-Fms proto-oncogene (macrophage colony stimulating A4b factor 1 (CSF-1) receptor) M84807 PDGFRa; platelet-derived growth factor alpha-receptor A4e U14173 Ski proto-oncogene A4g U18342 Sky proto-oncogene (Tyro3; Rse; Dtk) A4h Z50013 H-ras proto-oncogene; transforming G-protein A5c X13664 N-ras proto-oncogene; transforming G-protein A5e X81580 IGFBP-2; insulin-like growth factor binding protein 2; A5m autocrine and/or paracrine X66032 Cyclin B2 (G2/M-specific) A6d M83749 Cyclin D2 (G1/S-specific) A6g Z37110 Cyclin G (G2/M-specific) A6k U19596 pfBink4; cdk4 and cdk6 inhibitor A7c X56135 Prothymosin alpha A7m M36829 HSP84; heat shock 84 kD protein B1c M36830 HSP86; heat shock 86 kD protein B1d D78645 Glucose regulated protein, 78 kD; Grp78 B1m U34259 Golgi 4-transmembrane spanning transporter; MTP B2d L03529 C12r; coagulation factor II (thrombin) receptor B2j M83336 Interleukin-6 receptor beta chain; membrane glycoprotein B3c gp130 U36277 I-kappa B alpha chain B3m U06924 Stall; signal transducer and activator of transcription B4d L47850 Slat6; signal transducer and activator of transcription 6; B4g IL-4 Stal; STA6 S72408 Crk adaptor protein B4m U28423 Inhibitor of the RNA-activated protein kinase, 58-kDa B5i U10671 MAPK; MAP kinase; p38 B6m L02526 MAPKK1; MAP kinase kinase 3 (dual specilicity) B6a (MKK1) D11091 PI3-K p85; phosphatidylinositol 3-kinase regulatory B6h subunit; phosphoprotein p85; PKC-theta; protein kinase C theta type M60851 PDGF signaling pathway member 86k X95403 Rab-2 ras-related protein B7b U57311 14-3-3 protein ela B7g M21065 IRF1; Interferon regulatory factor 1 B7k X99063 Zyxin; LIM domain protein; alpha-actinin binding protein B7n U17162 BAG-1; bcl-2 binding protein with anti-cell death activity C1e U13705 Glutathione peroxidase (plasma protein); selenoprotein C1l X76341 Glutathione reductase C1m J03752 Glutathione S-transferase (microsomal) C2a J04696 Glutathione S-transferase Mu 1 C2b M94335 c-Akl proto-oncogene; Rac-alpha; protein kinase B C2k (PKB) U97076 FLIP-L; apoptosis inhibitor; FLICE-like inhibitory C3h protein L28177 Gadd45; growth arrest and DNA-damage-inducible C3j protein X68193 Nm23-M2; nucleoside diphosphate kinase B; metastasis- C4c reducing protein; c-myo-related transcription factor D83966 Protein tyrosine phosphatase C4g U25995 RIP cell death protein; Fas/APO-1 (CD95) interactor, C4j contains death domain U25844 SPI3; serpin; similar to human proteinase inhibitor 6 C4l (placental thrombin inhibitor) serine proteinase inhibitor M59376 Tumor necrosis factor receptor 1; TNFR-1 C5d X96618 PA6 stromal protein; RAG1 gene activator C6a X92410 MHR23B; Rad23 UV excision repair protein homologue; C6j xeroderma pigmentosum group C (XPC) repair complementing protein U17698 Abiphilln-1 (abl-1) similar to HOXD3 D1a L12721 Adipocyte differentation-associated protein D1c L36495 Basic domain/leucine zipper transcription factor D1e M58566 Butyrate response factor 1 D1i U36340 CACCC Box-binding protein BKLF D1j X72310 DP-1 (DRTF-polipeptide 1) cell cycle regulatory D2g transcription factor J04103 Ets-2 transcription factor D3b X55123 GATA-3 transcription factor D3f U20344 Gut-specific Kruppet-like factor GKLF D3i X53478 HMG-14 non histone chromosomal protein D3m J03168 Interferon regulatory factor 2 (IRF 2) |D4| D90176 NF-1B protein (transcription factor) D5f U20532 Nuclear factor related to P45 NF-E2 D5h U53228 Nuclear hormone receptor ROR-ALPHA-1 D5i U41626 Split hand/foot gene D5m M35523 Retinoic acid binding protein II cellular (CRABP-II) D6e U09419 Retinoid X receptor interacting protein (RIP 15) D6g X61753 Transcription factor 1 for heat shock gene D6i U51037 Transcription factor CTCF (11 zinc fingers) D6l X91753 Transcription factor SEF2 D7e X57621 YB1 DNA binding protein D7j L13968 YY1 (UCRBP) transcriptional factor D7k Z31663 Activin type I receptor E1a U56819 C-C chemokine receptor (Monocyte chemoattractant E1d protein 1 receptor (MCP-1RA) M98547 Growth factor receptor E2l X13358 Glucocorticoid receptor form A E3m L06039 CD31 (Platelet endothelial cell adhesion molecule 1) E6d M27129 CD44 antigen E6e U43512 Dystroglycan 1 E6m X04648 Glutamate receptor channel subunit gamma E6n X69902 Integrin alpha 6 E7d Y00769 Integrin beta E7g J02870 Lamimin receptor 1 E7j L24755 Bone morphogenetic protein 1 F1b X81584 Insulin-like growth factor binding protein-6 (IGFBP 6) F2i X81581 Insulin-like growth factor binding protein-3 (IGFBP-3) F2k X81582 Insulin-like growth factor binding protein-4 (IGFBP-4) F2l X81583 Insulin-like growth factor binding protein-5 (IGFBP-5) F2m X04480 Insulin-like growth factor-IA F3a X12531 Macrophage inflammatory protein F3e U60530 Mad related protein 2 (MADR2) F3h M14220 Neuroleukin F3m U22516 Placental ribonuclease inhibitor (Angiogenin) F4a M13177 Transforming growth factor beta F4f X57413 Transforming growth factor beta 2 F4g M13806 Cytoskeletal epidermal keratin (14 human) F5h M10937 Epidermal keratin (1 human) F5k U04443 Non-muscle myosin light chain 3 F6b X51438 Vimentin F6d J04946 Angiotensin-converting enzyme (ACE) (clone ACE.5.) F6f M14222 Cathepsin B F6g X53337 Cathepsin D F6h X06086 Cathepsin L F6j M12302 Cyotoxic T lymphocyte-specific serine protease CCP I F6m gene (CTLA-1) M55617 Masi cell protease (MMCP)-4 F7b X83536 Membrane type matrix metalloproteinase F7c X16490 Plaminogen activator inhibitor-2 F7i J05609 Serine protease inhibitor homolog J6 F7l L19622 TIMP-3 tissue inhibitor of metalloproteinases-3 F7n

TABLE 15 Differentially Expressed Genes in Healer and Non-Healer Dendritic Cells GENE NAME Array location 50% > Elk-1 ets related proto-oncogene A3a MRL > Pim-1 proto-oncogene A4a MRL > cyclin A A6a MRL > BRCA 1 A1b MRL > VHL A2b MRL > Fli-1 ets related proto-oncogene A3b MRL > BRCA2 A1c MRL > Fos-B A3c MRL > Dcc; netrin receptor A1d MRL > Fra-2 A3d MRL > EB1 APC binding protein A1e MRL > Gll oncogene A3e MRL > PDGFRa A4e MRL > N-ras A5e MRL > ezrin A1f B6 > B-myb proto-oncogene A2f MRL > Jun-B A3f MRL > Ret proto-oncogene A4f MRL > Shc transforming adaptor protein A5f MRL > Madr1 A1g MRL > c-ErbA oncogene A2g MRL > Skl proto-oncogene A4g MRL > CSF-1 A5g MRL > Mdm2 A1h MRL > c-Fos proto-oncogene A2h MRL > Sky proto-oncogene A4h MRL > Nf2; merlin A1i MRL > c-Jun proto-oncogene A2i MRL > p107; RBL1 A1j MRL > p130 A1k MRL > p53; tumor suppressor A1l MRL > Fyn proto-oncogene B5a B6 > Mapkk1 B6a B6 > Gem B7a B6 > Hck tyrosine protein kinase B5b B6 > Mapkk3 B6b B6> Rab-2 ras related protein B7b B6 > B7-2; T lymphocyte activation antigen CD86 B2g MRL > Mapkapk-2 B5n B6 > zyxln B7n B6 > Craf-1 C3c B6 > Nm23-M2 C4c B6 > DAD-1 C3d B6 > Nur77 early response protein C4d B6 > Faf1 C3e B6 > p55cdc C4e B6 > Fas1 receptor C3f B6 > PD-1 possible cell death inducer C4f B6 > Adenosine A2M2 receptor C2g B6 > Fas1 C3g B6 > protein tyrosine kphosphatase C4g B6 > Adenosine A3 receptor C2h B6 > MHR23A C6i B6 > Blk C2j B6 > c-Akt proto-oncogene C2k B6 > Glutathione peroxidase C1l B6 > iNOS1 C3m B6 > interleukln 1 receptor C3n B6 > stromolysin 3 C4n B6 > activating transcription factor 4 D1b MRL > Pax6 D6b B6 > SRY-box containing gene 4 D7b B6 > adipocyte differentiation-associated protein D1c MRL > transcription factor RelB D7c B6 > AT motif-binding factor ATBF1 D1d MRL > transcription factor NURR-1 D1g MRL > nuclear factor related to P45NF-E2 D5h B6 > Butyrate response factor 1 D1i MRL > CCAAT-binding transcription factor D1k MRL > IRF-2 D4l B6 > split hand/foot gene D5m B6 > Zinc finger transcription factor RU49 D7m B6 > SOX-3 D5n B6> Zinc finger X-chromosomal protein ZFX D7n B6 > activin type 1 receptor E1a B6 > orphan receptor E1b B6 > G-protein coupled receptor E5c B6> MCP-1RA E1d B6 > CD31 E6d B6 > CD4 receptor E1e B6 > GABA-A transporter 1 E5e B6 > CD44 E6e B6 > CD14 E6h B6 > CD22 E6i B6 > desmocollin 2 E6l B6 > interferon-gamma receptor E2m B6 > dystroglycan 1 E6m B6 > ERBB-3 receptor E1n B6 > interleukin-1 receptor type II E2n B6> growth hormone receptor E3n B6 > VLA-3 alpha subunit E7n B6> angiogenin F4a B6 > interleukin 15 F5a B6> KIF3B F6a B6 > non-muscle myosin light chain 3 F6b B6> prepro-endothelin-3 F4c B6 > TGF-beta 2 F4g B6 > TNF-beta F4h B6 > plasminogen activator inhibitor F7h B6 > IGFBP-6 F2i B6 > uromodulin F4i B6 > cathepsin H F6i B6 > plasminogen activator inhibitor 2 F7i B6 > vascular endothelial growth factor F4j B6 > cytoskeletal epidermal keratin 19 F5j B6 > cathepsin L F6j B6 > Spi-2 F7j B6 > interleukin-1 beta F4k B6 > collagenase type IV F6k B6 > serine protease inhibitor 2.4 F7k B6 > follistatin F1l B6> IGFBP-4 F2l B6 > interleukin 10 F4l B6 > cytotoxic cell protease (B10) F6l B6 > serine protease inhibitor homolog J6 F7l B6 > MIG F1m B6 > neuroleukin F3m B6 > interleukin 11 F4m B6 > KIF1A F5m B6 > CTLA1 F6m B6 > TIMP-2 F7m B6 > glial cell neuronal growth factor F1n B6 > IGF-2 F2n B6 > oncostatin M F3n B6 > Interleukin 12 (p40) beta chain F4n B6 > gelatinase B F6n B6 > TIMP-3 F7n B6 >

TABLE 16 Segregation of Quantitative Trait Loci in Phenotype Congenic Mice Marker 1-2-3-4-5 6-7-8-9-10 11-12-13- 14-15-17 1-185 (59) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 1-228 (72) B-B-B-B-B B-B B-B B B-B-B B-B-B 2-329 (45) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 2-207 (60) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 2-107 (76) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 2-113 (103) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 3-310 (38) B-B-B-B-B H-H-H-H-B B-B-B B-B-H 4-149 (0) H-H-H-H-H H-H-H-H-S S-S-S S-S- 4-236 (12) U-H- B-B-B 4-151 (29) H-S-B-U-H H-B-H-H-H H-S-S H-H-B 4-306 (51) U-B-H-H-H H-S-U S-S-B 4-127 (78) U-B-U-U-U U-U-B-U-B B-B-B B-B-B 5-255 (34) H-U-S-U-S S-S-H-H-S H-H-H S-S-B 5-215 (71) S-U-S-U-U U-S-U-S-H H-S-S S-S-B 7-228 (19) H-B-B-U-H B-B-H-B-H B-H-B B-B-B 7-220 (52) H-S-B-S-3 B-H-B-H-S B-S-S H-H-H 7-237 (53) H-S-B-H-B B-H-B-H-S H-S-S H-H-H 8-211 (49) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 8-166 B-B-B-B-B B-B-B-B-B B-B-B B-B-B 9-297 (15) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 9-207 (33) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 10-106 (17) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 10-198 (40) H-H-B-B-B B-H-H-H-S H-H-S H-H-B 10-233 (62) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 11-74 (0) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 11-235 (20) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 11-245 (43) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 11-48 (77) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 12-158 (38) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 12-132 (52) B-B-B-B-B B-B-B-B-B B-B-B H-H-B 12-233 (52) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 13-135 (10) B-B-B-B-B B-B-B-B-B H-H-H- B-H-H 13-115 (11) B-B-B-B-B B-B-B-B-B H-H-H B-H-H 13-116 (13) B-B-B-B-B B-B-B-B-B H-H-H B-H-H 13-60 (16) B-B-B-S-H B-S-S-H-B H-H-H B-H-H 13-245 (30) B-B-B-S-H B-U-S-H-B H-H-H B-H-H 13-139 (32) H-H-S-H-B B-S-S-H-H H-S-H S-S-H 13-NDS1 (32) H-H-S-H-B B-S-S-H-H H-S-H S-S-H 13-13 (35) H-H-S-H-B B-S-S-H-H H-S-H S-S-H 13-184 (35) H-H-S-H-B B-S-S-H-H H-S-H S-S-H 13-122 (36) H-H-S-U-B B-S-S-H-B H-S-H S-S-H 13-124 (37) H-H-S-U-B B-S-S-H-B H-S-H S-S-H 13-142 (37) H-H-S-H-B B-S-S-H-B H-S-H S-S-H 13-26 (38) H-H-S-H-B B-S-S-H-B H-S-H S-S-U 13-231 (39) H-H-S-B-B B-S-U-H-B H-S-H S-S-H 13-11 (40) H-H-S-U-B B-S-U-H-U H-S-H S-S-H 13-254 (40) H-H-S-H-B B-S-S-H-B H-S-H S-S-H 13-191 (45) H-H-S-U-B B-S-S-S-B H-S-H S-S-H 13-27 (42) H-H-S-U-B B-S-S-S-B B-H-H H-B-U 13-144 (48) H-H-S-H-B B-S-S-S-B B-H-H H-B-B 13-148 (59) B-B-B-B-B B-B-B-B-B B-B-B B-B-U 13-129 (60) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 13-260 (65) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 14-110 (3.5) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 14-121 (17) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 15-230 (28) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 15-189 (49) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 15-242 (56) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 15-244 (56) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 16-32 (2) B-B-B-B-B B-B-H-H-B B-B-B B-B-B 16-39 (29) S-H-H-S-H H-H-S-H-H B-H-H H-H-B 18-158 (16) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 18-123 (31) B-B-B-B-B B-B-B-B-B B-B-B B-B-B 19-101 (26) B-B-B-B-B B-B-B-B-B B-B-B B-B-B

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1. A method of identifying a factor involved in enhanced wound healing, comprising the steps of: contacting a mouse model of wound healing with a composition selected from the group consisting of: serum, a fraction of serum, and an extract of at least one healer mouse tissue, wherein the mouse model is a healer mouse, wherein a hole punched in an ear of a healer mouse will close at least 70% within 60 days; and assaying a biological property associated with wound healing in the mouse model, wherein a composition which enhances the biological property is identified as comprising a factor involved in enhanced wound healing.
 2. A method of screening test compounds for the ability to enhance wound healing, comprising the steps of: contacting a mouse model of wound healing with a test compound, wherein the mouse model is a healer mouse, wherein a hole punched in an ear of a healer mouse will close at least 70% within 60 days; and measuring the effect of the test compound on a biological property associated with wound healing in the mouse model, wherein a test compound which enhances the biological property is identified as a potential factor for enhancing wound healing.
 3. The method of claim 1 wherein the biological property is selected from the group consisting of enhanced wound healing, enhanced tissue regeneration, cell growth, apoptosis, cell movement, cell adhesion, cell replication, DNA synthesis, protein synthesis, mRNA synthesis, and mRNA stability.
 4. The method of claim 2 wherein the biological property is selected from the group consisting of enhanced wound healing, enhanced tissue regeneration, cell growth, apoptosis, cell movement, cell adhesion, cell replication, DNA synthesis, protein synthesis, mRNA synthesis, and mRNA stability.
 5. The method of claim 2 further comprising the step of comparing the effect of the test compound on the biological property of the healer mouse model with a biological property of a non-healer mouse model of wound healing.
 6. The method of claim 2 wherein the mouse model is an MRL mouse.
 7. The method of claim 2 wherein the mouse model is a healer mouse which is created by performing a cross between a mouse of a healer strain and a mouse of a non-healer strain and selecting an offspring with a healer phenotype.
 8. The method of claim 7 wherein the healer strain is MRL.
 9. The method of claim 7 wherein the non-healer strain is C57BL/6.
 10. The method of claim 2 wherein the mouse model is created by: performing a cross between a mouse of a healer strain and a mouse of a non-healer strain to form F₁ progeny mice; performing a cross between two of the F₁ progeny mice to form F2 progeny mice; and selecting F₂ progeny with a healer phenotype.
 11. The method of claim 2 wherein the mouse model is created by: (a) performing a cross between a mouse of a healer strain and a first mouse of a non-healer strain to form an F₁ mouse; and (b)performing a back-cross between the F₁ mouse and a second mouse of the non-healer strain; and (c) selecting an offspring with a healer phenotype.
 12. The method of claim 11, further comprising successive back-crossing between the mouse obtained in step (c) and a mouse of the non-healer strain.
 13. The method of claim 2 wherein the mouse model is created by: (a) performing a cross between a first mouse of a healer strain and a mouse of a non-healer strain to form an F₁ mouse; (b) performing a back-cross between the F₁ mouse and a second mouse of the healer strain; and (c) selecting an offspring with a healer phenotype.
 14. The method of claim 13, further comprising successive back-crossing between the mouse obtained in step (c) and a mouse of the healer strain.
 15. The method of claim 2 wherein the biological property is regeneration of a wounded tissue.
 16. The method of claim 15 wherein the wounded tissue is selected from the group consisting of spinal cord, optic nerve, cartilage, dermis, epidermis, blood vessels, and liver.
 17. The method of claim 2 wherein the mouse model is a male mouse.
 18. The method of claim 2 wherein the mouse model is a female mouse.
 19. The method of claim 2 wherein the mouse model is T cell deficient.
 20. The method of claim 2 further comprising the step of comparing the effect of the test compound on the biological property of the healer mouse model with a biological property of a non-healer mouse model of wound healing, wherein a test compound which alters the biological property of the non-healer mouse model to resemble the biological property of the healer mouse model is identified as a potential factor for enhancing wound healing.
 21. The method of claim 2 wherein the mouse model comprises one or more quantitative trait loci selected from the group consisting of heal1, heal2, heal3, heal4, and heal5.
 22. The method of claim 2 wherein the mouse model comprises a genotype of heal3^(S/S).
 23. The method of claim 2 wherein the mouse model comprises a genotype of heal1^(b/b) and heal3^(S/S). 